U.S. patent application number 10/211701 was filed with the patent office on 2003-03-06 for materials and methods for treating ocular-related disorders.
This patent application is currently assigned to GenVec, Inc.. Invention is credited to Brough, Douglas E., Kovesdi, Imre, McVey, Duncan L., Wei, Lisa.
Application Number | 20030045498 10/211701 |
Document ID | / |
Family ID | 33436589 |
Filed Date | 2003-03-06 |
United States Patent
Application |
20030045498 |
Kind Code |
A1 |
Kovesdi, Imre ; et
al. |
March 6, 2003 |
Materials and methods for treating ocular-related disorders
Abstract
The present invention is directed to a method of
prophylactically or therapeutically treating an animal for at least
one ocular-related disorder, e.g., ocular neovascularization or
age-related macular degeneration. The method comprises contacting
an ocular cell with an expression vector comprising a nucleic acid
sequence encoding an inhibitor of angiogenesis and the same or
different nucleic acid sequence encoding a neurotrophic agent. The
method also can comprise contacting an ocular cell with different
expression vectors, each comprising a nucleic acid sequence
encoding an inhibitor of angiogenesis and/or a nucleic acid
sequence encoding a neurotrophic agent. In addition, the present
invention provides a viral vector comprising a nucleic acid
sequence encoding pigment epithelium-derived factor (PEDF) or a
therapeutic fragment thereof.
Inventors: |
Kovesdi, Imre; (Rockville,
MD) ; Brough, Douglas E.; (Gaithersburg, MD) ;
Wei, Lisa; (Gaithersburg, MD) ; McVey, Duncan L.;
(Derwood, MD) |
Correspondence
Address: |
LEYDIG VOIT & MAYER, LTD
TWO PRUDENTIAL PLAZA, SUITE 4900
180 NORTH STETSON AVENUE
CHICAGO
IL
60601-6780
US
|
Assignee: |
GenVec, Inc.
65 West Watkins Mill Road
Gaithersburg
MD
20878
|
Family ID: |
33436589 |
Appl. No.: |
10/211701 |
Filed: |
August 2, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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10211701 |
Aug 2, 2002 |
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PCT/US01/04203 |
Feb 9, 2001 |
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10211701 |
Aug 2, 2002 |
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09599997 |
Jun 23, 2000 |
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60228337 |
Aug 28, 2000 |
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60181743 |
Feb 11, 2000 |
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60181743 |
Feb 11, 2000 |
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Current U.S.
Class: |
514/44A ;
435/320.1; 435/455 |
Current CPC
Class: |
C12N 15/85 20130101;
A61K 48/00 20130101; C07K 14/71 20130101; C07K 14/811 20130101;
A61P 27/02 20180101; A61K 48/005 20130101; C12N 2799/022 20130101;
C12N 2710/10343 20130101; A61K 48/0075 20130101; C12N 15/86
20130101 |
Class at
Publication: |
514/44 ;
435/320.1; 435/455 |
International
Class: |
A61K 048/00; C12N
015/85 |
Claims
What is claimed is:
1. A method of prophylactically or therapeutically treating an
animal for an ocular-related disorder, wherein the method comprises
contacting an ocular cell with (a) an expression vector comprising
a nucleic acid sequence encoding an inhibitor of angiogenesis and
the same or different nucleic acid sequence encoding a neurotrophic
agent, or (b) different expression vectors, each comprising a
nucleic acid sequence encoding an inhibitor of angiogenesis and/or
a nucleic acid sequence encoding a neurotrophic agent, such that
the nucleic acid sequence encoding the inhibitor of angiogenesis
and/or the nucleic acid sequence encoding the neurotrophic agent
are expressed, thereby resulting in the production of the inhibitor
of angiogenesis and the neurotrophic agent to prophylactically or
therapeutically treat the animal for an ocular-related
disorder.
2. The method of claim 1, wherein the method comprises contacting
the ocular cell with an expression vector comprising the nucleic
acid sequence encoding the inhibitor of angiogenesis and the
nucleic acid sequence encoding the neurotrophic agent.
3. The method of claim 2, wherein the nucleic acid sequence
encoding the inhibitor of angiogenesis and the nucleic acid
sequence encoding the neurotrophic agent are the same nucleic acid
sequence.
4. The method of claim 1, wherein the method comprises contacting
the ocular cell with at least two different types of expression
vectors, wherein each expression vector comprises a nucleic acid
sequence encoding an inhibitor of angiogenesis and/or a nucleic
acid sequence encoding a neurotrophic factor.
5. The method of claim 1, wherein the ocular-related disorder is
ocular neovascularization.
6. The method of claim 5, wherein the ocular neovascularization is
neovascularization of the choroid.
7. The method of claim 5, wherein the ocular neovascularization is
neovascularization of the retina.
8. The method of claim 7, wherein the neovascularization of the
retina is associated with diabetic retinopathy.
9. The method of claim 1, wherein the ocular-related disorder is
age-related macular degeneration.
10. The method of claim 1, wherein at least one expression vector
is an adeno-associated vector.
11. The method of claim 1, wherein at least one expression vector
is an adenoviral vector.
12. The method of claim 11, wherein at least one expression vector
is an adenoviral vector and at least one expression vector is an
adeno-associated viral vector.
13. The method of claim 11, wherein the adenoviral vector is
replication deficient.
14. The method of claim 1, wherein the expression vector(s) is
(are) administered to cells of neural origin, ciliary epithelial
cells, retinal pigment epithelial cells, glial cells, fibroblasts,
endothelial cells, or cells of the trabecular meshwork.
15. The method of claim 1, wherein the expression vector(s) is
(are) administered to iris epithelial cells, corneal cells, ciliary
epithelial cells, Mueller cells, or astrocytes.
16. The method of claim 1, wherein the expression vector(s) is
(are) administered to a patient greater than 55 years of age.
17. The method of claim 1, wherein the expression vector(s) is
(are) administered to an area of vascular leakage.
18. The method of claim 1, wherein the expression vector(s) is
present in or on a device that allows controlled release of the
expression vector(s).
19. The method of claim 1, wherein the expression vector(s) is
(are) administered topically, subconjunctivally, retrobulbarly,
periocularly, subretinally, suprachoroidally, or intraocularly.
20. The method of claim 4, wherein at least one expression vector
comprises the nucleic acid sequence encoding an inhibitor of
angiogenesis.
21. The method of claim 1, wherein the inhibitor of angiogenesis is
selected from the group consisting of an anti-angiogenic factor, an
anti-sense molecule specific for an angiogenic factor, a ribozyme,
and a receptor for an angiogenic factor.
22. The method of claim 1, wherein the nucleic acid sequence
encoding the inhibitor of angiogenesis encodes multiple inhibitors
of angiogenesis.
23. The method of claim 4, wherein at least one expression vector
comprises the nucleic acid sequence encoding a neurotrophic
factor.
24. The method of claim 4, wherein at least one expression vector
comprises the nucleic acid sequence encoding an inhibitor of
angiogenesis and the nucleic acid sequence encoding a neurotrophic
agent.
25. The method of claim 4, wherein the inhibitor of angiogenesis
and the neurotrophic agent are a single factor.
26. The method of claim 1, wherein the neurotrophic agent is
pigment epithelial-derived factor.
27. The method of claim 1, wherein the method comprises
administering the expression vector(s) in two or more applications
to the same eye of the animal.
Description
CROSS-REFERENCE TO RELATED PATENT APPLICATIONS
[0001] This patent application is a continuation of International
Patent Application No. PCTIUS01/04203, filed Feb. 9, 2001, which
designates the U.S., and which claims the benefit of U.S.
Provisional Patent Application No. 60/228,337, filed Aug. 28, 2000,
and which also claims the benefit of U.S. Provisional Patent
Application No. 60/181,743, filed Feb. 11, 2000, and which also is
a continuation-in-part of U.S. patent application Ser. No.
09/599,997, filed Jun. 23, 2000, which claims the benefit of U.S.
Provisional Patent Application No. 60/181,743, filed Feb. 11,
2000.
FIELD OF THE INVENTION
[0002] The present invention relates to a method of
prophylactically or therapeutically treating an ocular disorder as
well as materials useful for treating an ocular disorder.
BACKGROUND OF THE INVENTION
[0003] An overwhelming majority of the world's population will
experience some degree of vision loss in their lifetime. Vision
loss affects virtually all people regardless of age, race, economic
or social status, or geographical location. Ocular-related
disorders, while often not life threatening, necessitate life-style
changes that jeopardize the independence of the afflicted
individual. Vision impairment can result from most all ocular
disorders, including diabetic retinopathies, proliferative
retinopathies, retinal detachment, toxic retinopathies, retinal
vascular diseases, retinal degenerations, vascular anomalies,
age-related macular degeneration and other acquired disorders,
infectious diseases, inflammatory diseases, ocular ischemia,
pregnancy-related disorders, retinal tumors, choroidal tumors,
choroidal disorders, vitreous disorders, trauma, cataract
complications, dry eye, and inflammatory optic neuropathies.
[0004] Leading causes of severe vision loss and blindness are
ocular-related disorders wherein the vasculature of the eye is
damaged or insufficiently regulated. Ocular-related diseases
comprising a neovascularization aspect are many and include, for
example, exudative age-related macular degeneration, diabetic
retinopathy, corneal neovascularization, choroidal
neovascularization, neovascular glaucoma, cyclitis, Hippel-Lindau
Disease, retinopathy of prematurity, pterygium, histoplasmosis,
iris neovascularization, macular edema, glaucoma-associated
neovascularization, and the like. It is likely that severe vision
loss does not result directly from neovascularization, but the
effect of neovascularization on the retina. The retina is a
delicate ocular membrane on which images are received. Near the
center of the retina is the macula lutea, an oval area of retinal
tissue where visual sense is most acute. The retina is most
delicate at the fovea centralis, the central depression located in
the center of the macula. Damage of the retina, i.e., retinal
detachment, retinal tears, or retinal degeneration, is directly
connected to vision loss. While a common cause of retinal
detachment, retinal tears, and retinal degeneration is abnormal,
i.e., uncontrolled, vascularization of various ocular tissues, this
is not always the case. Atrophic complications associated with
age-related macular degeneration, nonproliferative diabetic
retinopathy, and inflammatory ocular damage are not associated with
neovascularization, but can result in severe vision loss if not
treated. Disorders associated with both neovascular and atrophic
components, such as age-related macular degeneration and diabetic
retinopathy, are particularly difficult to treat due to the
emergence of a wide variety of complications.
[0005] Age-related macular degeneration is the leading cause of
blindness in patients over 65 years of age. As the elderly
population of the world increases, the incidence of age-related
macular degeneration is expected to increase dramatically, reaching
a predicted 7.5 million cases in the United States alone by the
year 2030 (Hyman et al., Am. J Epidemiol, 118, 213-227 (1983)).
Age-related macular degeneration (AMD) is a progressive,
degenerative disorder of the eye resulting initially in loss of
visual acuity. Complications arising with advanced age-related
macular degeneration include atrophic and exudative complications.
Atrophic complications stem from retinal pigment epithelial cell
loss resulting in atrophy of the retinal pigment epithelium (RPE).
Exudative complications include disciform scars (i.e., scarring
involving fibrous elements) and neovascularization. Severe vision
loss occurs as neovascularization or atrophy disturbs the foveal
center (Bressler et al., Ophthalmology, 102, 1206-1211 (1995)).
Ultimately, blindness from age-related macular degeneration stems
from degeneration of the RPE and the subsequent death of
photoreceptors.
[0006] Similarly, diabetic retinopathy is subdivided into a
nonproliferative stage, which typically occurs first, and a
proliferative stage. Vision loss associated with nonproliferative
diabetic retinopathy stems from retinal edema, in particular
diabetic macular edema, resulting from vascular leakage. Focal and
diffuse vascular leakage occurs as a result of microvascular
abnormalities, intraretinal microaneurysms, capillary closure, and
retinal hemorrhages. Prolonged periods of vascular leakage
ultimately lead to thickening of the basement membrane and
formation of soft and hard exudates. Nonproliferative diabetic
retinopathy is also characterized by loss of retinal pericytes. The
proliferative stage of diabetic retinopathy is characterized by
neovascularization and fibrovascular growth (i.e., scarring
involving glial and fibrous elements) from the retina or optic
nerve over the inner surface of the retina or disc or into the
vitreous cavity. Retinal neovascularization is the leading cause of
vision loss associated with proliferative diabetic retinopathy.
[0007] For many ocular-related disorders, no efficient therapeutic
options currently are available. Laser photocoagulation involves
administering laser burns to various areas of the eye and is used
in the treatment of many neovascularization-linked disorders. For
example, focal macular photocoagulation is used to treat areas of
vascular leakage in the macula (Murphy, Amer. Family Physician,
51(4), 785-796 (1995)). Similarly, neovascularization, in
particular, advanced proliferative retinopathy, is commonly treated
with scatter or panretinal photocoagulation. However, laser
treatment may cause permanent blind spots corresponding to the
treated areas. Laser treatment may also cause persistent or
recurrent hemorrhage, increase the risk of retinal detachment, or
induce neovascularization or fibrosis. With respect to age-related
macular degeneration, many patients eventually experience severe
vision loss in spite of treatment. Other treatment options for
ocular-related disorders include thermotherapy, radiation therapy,
surgery, e.g., macular translocation, removal of excess ocular
tissue, and the like. However, in most cases, all available
treatment options have limited therapeutic effect, require
repeated, costly procedures, and/or are associated with dangerous
side-effects.
[0008] Given the prevalence of ocular-related disorders, there
remains a need for an effective prophylactic and therapeutic
treatment of ocular-related disorders, in particular those
ocular-related disorders associated with both atrophic and
neovascular complications, such as diabetic retinopathy and
age-related macular degeneration. Accordingly, the present
invention provides materials and methods for prophylactically and
therapeutically treating ocular-related disorders, including
treatment of nonproliferative complications and proliferative
complications. This and other advantages of the present invention
will become apparent from the detailed description provided
herein.
BRIEF SUMMARY OF THE INVENTION
[0009] The present invention provides a method for the prophylactic
or therapeutic treatment of ocular-related disorders. In
particular, the present invention provides a method of
prophylactically or therapeutically treating an animal for at least
one ocular-related disorder, such as ocular neovascularization and
age-related macular degeneration. The method comprises contacting
an ocular cell with (a) an expression vector comprising a nucleic
acid sequence encoding an inhibitor of angiogenesis and the same or
different nucleic acid sequence encoding a neurotrophic agent, or
(b) different expression vectors, each comprising a nucleic acid
sequence encoding an inhibitor of angiogenesis and/or a
neurotrophic agent. The nucleic acid sequence encoding the
inhibitor of angiogenesis and/or the nucleic acid sequence encoding
the neurotrophic agent is (are) expressed, thereby resulting in the
production of the inhibitor of angiogenesis and/or the neurotrophic
agent to prophylactically or therapeutically treat the animal for
an ocular-related disorder.
[0010] Preferably, the nucleic acid sequence encoding the inhibitor
of angiogenesis and the nucleic acid sequence encoding the
neurotrophic agent are the same nucleic acid sequence. More
preferably, the nucleic acid sequence encodes a factor comprising
both anti-angiogenic and neurotrophic activity. Most preferably,
the factor is PEDF.
[0011] In addition, the present invention provides a viral vector
comprising a nucleic acid sequence encoding pigment
epithelium-derived factor (PEDF) or a therapeutic fragment thereof
The nucleic acid sequence is operably linked to regulatory
sequences necessary for expression of PEDF or a therapeutic
fragment thereof. Preferably, the viral vector is an adenoviral
vector or an adeno-associated viral vector. Also preferably, the
viral vector further comprises one or more additional nucleic acid
sequences encoding therapeutic substances other than PEDF or a
therapeutic fragment thereof
DETAILED DESCRIPTION OF THE INVENTION
[0012] The present invention is directed to methods of
prophylactically or therapeutically treating an animal, preferably
a human, for at least one ocular-related disorder. The present
invention also provides materials for treating ocular-related
disorders. Ocular-related disorders appropriate for treatment using
the present inventive materials and methods include, but are not
limited to, diabetic retinopathies, proliferative retinopathies,
retinopathy of prematurity, retinal vascular diseases, vascular
anomalies, age-related macular degeneration and other acquired
disorders, endophthalmitis, infectious diseases, inflammatory
diseases, AIDS-related disorders, ocular ischemia syndrome,
pregnancy-related disorders, peripheral retinal degenerations,
retinal degenerations, toxic retinopathies, cataracts, retinal
tumors, corneal neovascularization, choroidal tumors, choroidal
disorders, choroidal neovascularization, neovascular glaucoma,
vitreous disorders, retinal detachment and proliferative
vitreoretinopathy, cyclitis, non-penetrating trauma, penetrating
trauma, post-cataract complications, Hippel-Lindau Disease, dry
eye, inflammatory optic neuropathies, macular edema, pterygium,
iris neovascularization, surgical-induced disorders, and the
like.
[0013] In particular, the present invention provides a method of
prophylactically or therapeutically treating an animal for at least
one ocular-related disorder, such as ocular neovascularization. The
method comprises contacting an ocular cell with an expression
vector comprising a nucleic acid sequence encoding at least one
inhibitor of angiogenesis and/or at least one neurotrophic agent.
Preferably, the method comprises contacting an ocular cell with an
expression vector comprising a nucleic acid sequence encoding an
inhibitor of angiogenesis and the same or different nucleic acid
sequence encoding a neurotrophic agent. Desirably, the nucleic acid
sequence encodes at least one inhibitor of angiogenesis and at
least one neurotrophic agent. The ocular neovascularization treated
by the present inventive method can be neovascularization of the
choroid. The choroid is a thin, vascular membrane located under the
retina. Abnormal neovascularization of the choroid results from,
for example, photocoagulation, anterior ischemic optic neuropathy,
Best's disease, choroidal hemangioma, metallic intraocular foreign
body, choroidal nonperfusion, choroidal osteomas, choroidal
rupture, bacterial endocarditis, choroideremia, chronic retinal
detachment, drusen, deposit of metabolic waste material, endogenous
Candida endophthalmitis, neovascularization at ora serrata,
operating microscope burn, punctate inner choroidopathy, radiation
retinopathy, retinal cryoinjury, retinitis pigmentosa,
retinochoroidal coloboma, rubella, subretinal fluid drainage,
tilted disc syndrome, Taxoplasma retinochoroiditis, tuberculosis,
and the like.
[0014] Neovascularization of the cornea is also appropriate for
treatment by the method of the present invention. The cornea is a
projecting, transparent section of the fibrous tunic, the outer
most layer of the eye. The outermost layer of the cornea contacts
the conjunctiva, while the innermost layer comprises the
endothelium of the anterior chamber. Corneal neovascularization
stems from, for example, ocular injury, surgery, infection,
improper wearing of contact lenses, and diseases such as, for
example, corneal dystrophies.
[0015] Alternatively, the ocular neovascularization is preferably
neovascularization of the retina. Retinal neovascularization is an
indication associated with numerous ocular diseases and disorders,
many of which are named above. Preferably, the neovascularization
of the retina treated in accordance with the present inventive
method is associated with diabetic retinopathy. Common causes of
retinal neovascularization include ischemia, viral infection, and
retinal damage. Neovascularization of the retina can lead to
macular edema, subretinal discoloration, scarring, and the like.
Complications associated with retina neovascularization stem from
breakage and leakage of newly formed blood vessels. Vision is
impaired as blood fills the vitreous cavity and is not efficiently
removed. Not only is the passage of light impeded, but an
inflammatory response to the excess blood and metabolites can cause
further damage to ocular tissue. In addition, the new vessels form
fibrous scar tissue, which, over time, will disturb the retina
causing retinal tears and detachment.
[0016] The present invention also provides a method for
prophylactically or therapeutically treating an animal for
age-related macular degeneration. The method comprises contacting
an ocular cell associated with age-related macular degeneration
with an expression vector comprising a nucleic acid sequence
encoding at least one inhibitor of angiogenesis and/or at least one
neurotrophic factor. Desirably, the expression vector comprises a
nucleic acid sequence encoding an inhibitor of angiogenesis and a
nucleic acid sequence encoding a neurotrophic agent. More
desirably, the nucleic acid sequence encoding the inhibitor of
angiogenesis and the nucleic acid sequence encoding the
neurotrophic agent are the same nucleic acid sequence. Preferably,
the age-related macular degeneration is associated with at least
one exudative complication. Exudative complications include, for
example, disciform scars (i.e., scarring involving fibrous
elements) and neovascularization. Alternatively, the age-related
macular degeneration is associated with at least one atrophic
complication. Atrophic complications include, for instance, the
formation of drusen and basal laminar deposits, irregularity of
retinal pigmentation, and accumulation of lipofuscin granules.
[0017] By "prophylactic" is meant the protection, in whole or in
part, against ocularrelated disorders, in particular ocular
neovascularization or age-related macular degeneration. By
"therapeutic" is meant the amelioration of the ocular-related
disorder, itself, and the protection, in whole or in part, against
further ocular-related disease, in particular ocular
neovascularization or age-related macular degeneration. One of
ordinary skill in the art will appreciate that any degree of
protection from, or amelioration of, an ocular-related disorder is
beneficial to a patient. The present invention is particularly
advantageous in that a therapeutic agent can be directly applied to
affected areas without the harmful side effects of presently
employed therapies.
[0018] The present inventive methods are useful in the treatment of
both acute and persistent, progressive ocular-related disorders.
For acute ailments, the expression vector comprising a nucleic acid
sequence encoding at least one inhibitor of angiogenesis and/or at
least one neurotrophic factor can be administered using a single or
multiple applications within a short time period. For persistent
ocular-related diseases, such as age-related macular degeneration
and diabetic retinopathy, numerous applications of the expression
vector may be necessary to realize a therapeutic effect.
[0019] One of ordinary skill in the art will appreciate that any of
a number of expression vectors known in the art are suitable for
use in the present inventive methods. Examples of suitable
expression vectors include, for instance, plasmids,
plasmid-liposome complexes, and viral vectors, e.g.,
parvoviral-based vectors (i.e., adeno-associated virus (AAV)-based
vectors), retroviral vectors, herpes simplex virus (HSV)-based
vectors, AAV-adenoviral chimeric vectors, and adenovirus-based
vectors. Any of these expression vectors can be prepared using
standard recombinant DNA techniques described in, e.g., Sambrook et
al., Molecular Cloning, a Laboratory Manual, 2d edition, Cold
Spring Harbor Press, Cold Spring Harbor, N.Y. (1989), and Ausubel
et al., Current Protocols in Molecular Biology, Greene Publishing
Associates and John Wiley & Sons, New York, N.Y. (1994).
[0020] Plasmids, genetically engineered circular double-stranded
DNA molecules, can be designed to contain an expression cassette
for delivery of the nucleic acid sequence encoding at least one
inhibitor of angiogenesis and/or at least one neurotrophic factor
to an ocular cell. Although plasmids were the first vector
described for administration of therapeutic nucleic acids, the
level of transfection efficiency is poor compared with other
techniques. By complexing the plasmid with liposomes, the
efficiency of gene transfer in general is improved. While the
liposomes used for plasmid-mediated gene transfer strategies have
various compositions, they are typically synthetic cationic lipids.
Advantages of plasmid-liposome complexes include their ability to
transfer large pieces of DNA encoding a therapeutic nucleic acid
and their relatively low immunogenicity.
[0021] Plasmids are often used for short-term expression. However,
a plasmid construct can be modified to obtain prolonged expression.
It has recently been discovered that the inverted terminal repeats
(ITR) of parvovirus, in particular adeno-associated virus (AAV),
are responsible for the high-level persistent nucleic acid
expression often associated with AAV (see, for example, U.S. Pat.
No. 6,165,754). Accordingly, the expression vector can be a plasmid
comprising native parvovirus ITRs to obtain prolonged and
substantial expression of at least one inhibitor of angiogenesis
and/or at least one neurotrophic factor. While plasmids are
suitable for use in the present inventive methods, preferably the
expression vector is a viral vector.
[0022] AAV vectors are viral vectors of particular interest for use
in gene therapy protocols. AAV is a DNA virus, which is not known
to cause human disease. AAV requires co-infection with a helper
virus (i.e., an adenovirus or a herpes virus), or expression of
helper genes, for efficient replication. AAV vectors used for
administration of a therapeutic nucleic acid have approximately 96%
of the parental genome deleted, such that only the terminal repeats
(ITRs), which contain recognition signals for DNA replication and
packaging, remain. This eliminates immunologic or toxic side
effects due to expression of viral genes. In addition, delivering
the AAV rep protein enables integration of the AAV vector
comprising AAV ITRs into a specific region of genome, if desired.
Host cells comprising an integrated AAV genome show no change in
cell growth or morphology (see, for example, U. S. Pat. No.
4,797,368). Although efficient, the need for helper virus or helper
genes can be an obstacle for widespread use of this vector.
[0023] Retrovirus is an RNA virus capable of infecting a wide
variety of host cells. Upon infection, the retroviral genome
integrates into the genome of its host cell and is replicated along
with host cell DNA, thereby constantly producing viral RNA and any
nucleic acid sequence incorporated into the retroviral genome. When
employing pathogenic retroviruses, e.g., human immunodeficiency
virus (HIV) or human T-cell lymphotrophic viruses (HTLV), care must
be taken in altering the viral genomic to eliminate toxicity. A
retroviral vector can additionally be manipulated to render the
virus replication-incompetent. As such, retroviral vectors are
thought to be particularly useful for stable gene transfer in vivo.
Lentiviral vectors, such as HIV-based vectors, are exemplary of
retroviral vectors used for gene delivery. Unlike other
retroviruses, HIV-based vectors are known to incorporate their
passenger genes into non-dividing cells and, therefore, can be of
use in treating atrophic forms of ocular-related disease.
[0024] HSV-based viral vectors are suitable for use as an
expression vector to introduce nucleic acids into ocular cells. The
mature HSV virion consists of an enveloped icosahedral capsid with
a viral genome consisting of a linear double-stranded DNA molecule
that is 152 kb. Most replication-deficient HSV vectors contain a
deletion to remove one or more intermediate-early genes to prevent
replication. Advantages of the herpes vector are its ability to
enter a latent stage that can result in long-term DNA expression,
and its large viral DNA genome that can accommodate exogenous DNA
up to 25 kb. Of course, this ability is also a disadvantage in
terms of short-term treatment regimens. For a description of
HSV-based vectors appropriate for use in the present inventive
methods, see, for example, U.S. Pat. Nos. 5,837,532; 5,846,782;
5,849,572; and 5,804,413 and International Patent Applications WO
91/02788, WO 96/04394, WO 98/15637, and WO 99/06583.
[0025] Adenovirus (Ad) is a 36 kb double-stranded DNA virus that
efficiently transfers DNA in vivo to a variety of different target
cell types. For use in the present inventive methods, the virus is
preferably made replication deficient by deleting select genes
required for viral replication. The expendable E3 region is also
frequently deleted to allow additional room for a larger DNA
insert. The vector can be produced in high titers and can
efficiently transfer DNA to replicating and non-replicating cells.
The newly transferred genetic information remains epi-chromosomal,
thus eliminating the risks of random insertional mutagenesis and
permanent alteration of the genotype of the target cell. However,
if desired, the integrative properties of AAV can be conferred to
adenovirus by constructing an AAV-Ad chimeric vector. For example,
the AAV ITRs and nucleic acid encoding the Rep protein incorporated
into an adenoviral vector enables the adenoviral vector to
integrate into a mammalian cell genome. Therefore, AAV-Ad chimeric
vectors are an interesting option for use in the present
invention.
[0026] Preferably, the expression vector of the present inventive
methods is a viral vector; more preferably, the expression vector
is an adenoviral vector. In the context of the present invention,
the adenoviral vector can be derived from any serotype of
adenovirus. Adenoviral stocks that can be employed as a source of
adenovirus can be amplified from the adenoviral serotypes 1 through
51, which are currently available from the American Type Culture
Collection (ATCC, Manassas, Va.), or from any other serotype of
adenovirus available from any other source. For instance, an
adenovirus can be of subgroup A (e.g., serotypes 12, 18, and 31),
subgroup B (e.g., serotypes 3, 7, 11, 14, 16, 21, 34, and 35),
subgroup C (e.g., serotypes 1, 2, 5, and 6), subgroup D (e.g.,
serotypes 8, 9, 10, 13, 15, 17, 19, 20, 22-30, 32, 33, 36-39, and
42-47), subgroup E (serotype 4), subgroup F (serotypes 40 and 41),
or any other adenoviral serotype. Preferably, however, an
adenovirus is of serotype 2, 5 or 9. However, non-group C
adenoviruses can be used to prepare replication-deficient
adenoviral gene transfer vectors for delivery of anti-angiogenic
factors and/or neurotrophic factors to ocular cells. Preferred
adenoviruses used in the construction of non-group C adenoviral
gene transfer vectors include Ad12 (group A), Ad7 (group B), Ad30
and Ad36 (group D), Ad4 (group E), and Ad41 (group F). Non-group C
adenoviral vectors, methods of producing non-group C adenoviral
vectors, and methods of using non-group C adenoviral vectors are
disclosed in, for example, U.S. Pat. Nos. 5,801,030; 5,837,511; and
5,849,561 and International Patent Applications WO 97/12986 and WO
98/53087.
[0027] The adenoviral vector is preferably deficient in at least
one gene function required for viral replication, thereby resulting
in a "replication-deficient" adenoviral vector. Preferably, the
adenoviral vector is deficient in at least one essential gene
function of the E1 region of the adenoviral genome required for
viral replication. In addition to a deficiency in the E1 region,
the recombinant adenovirus can also have a mutation in the major
late promoter (MLP). The mutation in the MLP can be in any of the
MLP control elements such that it alters the responsiveness of the
promoter, as discussed in International Patent Application WO
00/00628. More preferably, the vector is deficient in at least one
essential gene function of the E1 region and at least part of the
E3 region (e.g., an Xba I deletion of the E3 region). With respect
to the E1 region, the adenoviral vector can be deficient in at
least part of the E1a region and at least part of the E1b region.
Preferably, the adenoviral vector is "multiply deficient," meaning
that the adenoviral vector is deficient in one or more essential
gene functions required for viral replication in each of two or
more regions. For example, the aforementioned E1-deficient or E1-,
E3-deficient adenoviral vectors can be further deficient in at
least one essential gene of the E4 region. Adenoviral vectors
deleted of the entire E4 region can elicit lower host immune
responses.
[0028] Alternatively, the adenoviral vector lacks all or part of
the E1 region and all or part of the E2 region. However, adenoviral
vectors lacking all or part of the E1 region, all or part of the E2
region, and all or part of the E3 region also are contemplated
herein. In one embodiment, the adenoviral vector lacks all or part
of the E1 region, all or part of the E2 region, all or part of the
E3 region, and all or part of the E4 region. Suitable
replication-deficient adenoviral vectors are disclosed in U.S. Pat.
Nos. 5,851,806 and 5,994,106 and International Patent Applications
WO 95/34671 and WO 97/21826. For example, suitable
replication-deficient adenoviral vectors include those with at
least a partial deletion of the E1a region, at least a partial
deletion of the E1b region, at least a partial deletion of the E2a
region, and at least a partial deletion of the E3 region.
Alternatively, the replication-deficient adenoviral vector can have
at least a partial deletion of the E1 region, at least a partial
deletion of the E3 region, and at least a partial deletion of the
E4 region. Such multiply-deficient viral vectors are particularly
useful in that such vectors can accept large inserts of exogenous
DNA. Indeed, adenoviral amplicons, an example of a
multiply-deficient adenoviral vector which comprises only those
genomic sequences required for packaging and replication of the
viral genome, can accept inserts of approximately 36 kb.
[0029] Therefore, in a preferred embodiment, the expression vector
of the present inventive method is a multiply-deficient adenoviral
vector lacking all or part of the E1 region, all or part of the E3
region, all or part of the E4 region, and, optionally, all or part
of the E2 region. In this regard, it has been observed that an at
least E4-deficient adenoviral vector expresses a transgene at high
levels for a limited amount of time in vivo and that persistence of
expression of a transgene in an at least E4-deficient adenoviral
vector can be modulated through the action of a trans-acting
factor, such as HSV ICPO, Ad pTP, CMV-IE2, CMV-IE86, HIV tat,
HTLV-tax, HBV-X, AAV Rep 78, the cellular factor from the U205
osteosarcoma cell line that functions like HSV ICPO, or the
cellular factor in PC12 cells that is induced by nerve growth
factor, among others. In view of the above, the multiply deficient
adenoviral vector (e.g., the at least E4-deficient adenoviral
vector) preferably further comprises a nucleic acid sequence
encoding a trans-acting factor that modulates the persistence of
expression of the nucleic acid sequence encoding at least one
inhibitor of angiogenesis and/or at least one neurotrophic factor.
Alternatively, the ocular cell is contacted with a second
expression vector comprising a nucleic acid sequence encoding a
trans-acting factor that modulates the persistence of expression of
the nucleic acid sequence encoding at least one inhibitor of
angiogenesis and/or at least one neurotrophic factor. Preferably,
the nucleic acid sequence encoding the trans-acting factor does not
encode an adenoviral E4 region gene product. Whether expressed from
the adenoviral vector or supplied by a second expression vector,
preferably, the trans-acting factor is the Herpes simplex infected
cell polypeptide 0 (HSV ICPO).
[0030] It should be appreciated that the deletion of different
regions of the adenoviral vector can alter the immune response of
the mammal. In particular, deletion of different regions can reduce
the inflammatory response generated by the adenoviral vector.
Furthermore, the adenoviral vector's coat protein can be modified
so as to decrease the adenoviral vector's ability or inability to
be recognized by a neutralizing antibody directed against the
wild-type coat protein, as described in International Patent
Application WO 98/40509. Such modifications are useful for
long-term treatment of persistent ocular disorders.
[0031] Similarly, the coat protein of a viral vector, preferably an
adenoviral vector, can be manipulated to alter the binding
specificity or recognition of a virus for a viral receptor on a
potential host cell. For adenovirus, such manipulations can include
deletion of regions of the fiber, penton, or hexon, insertions of
various native or non-native ligands into portions of the coat
protein, and the like. Manipulation of the coat protein can broaden
the range of cells infected by a viral vector or enable targeting
of a viral vector to a specific cell type. For example, in one
embodiment, the expression vector is a viral vector comprising a
chimeric coat protein (e.g., a fiber, hexon pIX, pIIIa, or penton
protein), which differs from the wild-type (i.e., native) coat
protein by the introduction of a nonnative amino acid sequence,
preferably at or near the carboxyl terminus. Preferably, the
nonnative amino acid sequence is inserted into or in place of an
internal coat protein sequence. One of ordinary skill in the art
will understand that the nonnative amino acid sequence can be
inserted within the internal coat protein sequence or at the end of
the internal coat protein sequence. The resultant chimeric viral
coat protein is able to direct entry into cells of the viral, i.e.,
adenoviral, vector comprising the coat protein that is more
efficient than entry into cells of a vector that is identical
except for comprising a wild-type viral coat protein rather than
the chimeric viral coat protein. Preferably, the chimeric virus
coat protein binds a novel endogenous binding site present on the
cell surface that is not recognized, or is poorly recognized by a
vector comprising a wild-type coat protein. One direct result of
this increased efficiency of entry is that the virus, preferably,
the adenovirus, can bind to and enter numerous cell types which a
virus comprising wild-type coat protein typically cannot enter or
can enter with only a low efficiency.
[0032] In another embodiment of the present invention, the
expression vector is a viral vector comprising a chimeric virus
coat protein not selective for a specific type of eukaryotic cell.
The chimeric coat protein differs from the wild-type coat protein
by an insertion of a nonnative amino acid sequence into or in place
of an internal coat protein sequence. In this embodiment, the
chimeric virus coat protein efficiently binds to a broader range of
eukaryotic cells than a wild-type virus coat, such as described in
International Patent Application WO 97/20051.
[0033] Specificity of binding of an adenovirus to a given cell can
also be adjusted by use of an adenovirus comprising a short-shafted
adenoviral fiber gene, as discussed in U.S. Pat. No. 5,962,311. Use
of an adenovirus comprising a short-shafted adenoviral fiber gene
reduces the level or efficiency of adenoviral fiber binding to its
cell-surface receptor and increases adenoviral penton base binding
to its cell-surface receptor, thereby increasing the specificity of
binding of the adenovirus to a given cell. Alternatively, use of an
adenovirus comprising a short-shafted fiber enables targeting of
the adenovirus to a desired cell-surface receptor by the
introduction of a nonnative amino acid sequence either into the
penton base or the fiber knob.
[0034] Of course, the ability of a viral vector to recognize a
potential host cell can be modulated without genetic manipulation
of the coat protein. For instance, complexing an adenovirus with a
bispecific molecule comprising a penton base-binding domain and a
domain that selectively binds a particular cell surface binding
site enables one of ordinary skill in the art to target the vector
to a particular cell type.
[0035] Suitable modifications to a viral vector, specifically an
adenoviral vector, are described in U.S. Pat. Nos. 5,559,099;
5,731,190; 5,712,136; 5,770,442; 5,846,782; 5,926,311; 5,965,541;
6,057,155; 6,127,525; and 6,153,435 and International Patent
Applications WO 96/07734, WO 96/26281, WO 97/20051, WO 98/07865, WO
98/07877, WO 98/54346, and WO 00/15823. Similarly, it will be
appreciated that numerous expression vectors are available
commercially. Construction of expression vectors is well understood
in the art. Adenoviral vectors can be constructed and/or purified
using the methods set forth, for example, in U.S. Pat. No.
5,965,358 and International Patent Applications WO 98/56937, WO
99/15686, and WO 99/54441. Adeno-associated viral vectors can be
constructed and/or purified using the methods set forth, for
example, in U.S. Pat. No. 4,797,368 and Laughlin et al., Gene, 23,
65-73 (1983).
[0036] The selection of expression vector for use in the present
inventive method will depend on a variety of factors such as, for
example, the host, immunogenicity of the vector, the desired
duration of protein production, and the like. As each type of
expression vector has distinct properties, a researcher has the
freedom to tailor the present inventive method to any particular
situation. Moreover, more than one type of expression vector can be
used to deliver the nucleic acid sequence to the ocular cell. Thus,
the present invention provides a method of prophylactically or
therapeutically treating an animal for at least one ocularrelated
disorder, wherein the method comprises contacting an ocular cell
with different expression vectors, each comprising a nucleic acid
sequence encoding an inhibitor of angiogenesis and/or a nucleic
acid sequence encoding a neurotrophic agent. The nucleic acid
sequence encoding the inhibitor of angiogenesis and/or the nucleic
acid sequence encoding the neurotrophic agent are expressed,
thereby resulting in the production of the inhibitor of
angiogenesis and/or the neurotrophic agent to prophylactically or
therapeutically treat the animal for an ocular-related
disorder.
[0037] Preferably, at least two different types of expression
vector (i.e., a plasmid and a viral vector or two different viral
vectors) are delivered to the ocular cell. At least one expression
vector can comprise a nucleic acid sequence encoding an inhibitor
of angiogenesis. Similarly, at least one expression vector can
comprise a nucleic acid sequence encoding a neurotrophic agent.
Indeed, a mixture of expression vectors, some comprising the coding
sequence for an inhibitor of angiogenesis and some comprising the
coding sequence for a neurotrophic agent, can be administered.
Desirably, at least one expression vector comprises the nucleic
acid sequence encoding the inhibitor of angiogenesis and the
nucleic acid sequence encoding the neurotrophic agent. More
preferably, the nucleic acid sequence encoding the inhibitor of
angiogenesis and the nucleic acid sequence encoding the
neurotrophic agent are the same nucleic acid. Also preferably, the
inhibitor of angiogenesis and the neurotrophic agent are a single
factor. Preferably, the ocular cell is contacted with an adenoviral
vector and an adeno-associated viral vector. One of ordinary skill
in the art will appreciate the ability to capitalize on the
advantageous properties of multiple delivery systems to treat or
study ocular-related disorders.
[0038] One embodiment of the present invention provides a method
for prophylactically or therapeutically treating an animal for
age-related macular degeneration. The method comprises contacting
an ocular cell associated with age-related macular degeneration
with an expression vector comprising a nucleic acid sequence
encoding at least one inhibitor of angiogenesis and/or at least one
neurotrophic factor. Alternatively, the ocular cell is contacted
with at least two different types of expression vector, each
expression vector comprising a nucleic acid sequence encoding at
least one inhibitor of angiogenesis and/or at least one
neurotrophic factor. As age-related macular degeneration commonly
afflicts the elderly, preferably the expression vector is
administered to an animal, i.e., a human, at least 55 years old.
Age-related macular degeneration is a complex disease associated
with a wide variety of complications affecting a number of ocular
tissues. Ocular cells associated with age-related macular
degeneration include, but are not limited to, cells of neural
origin, cells of all layers of the retina, especially retinal
pigment epithelial cells, glial cells, and pericytes. Other ocular
cells that are suitable for use in the method of the present
invention include, for example, endothelial cells, iris epithelial
cells, corneal cells, ciliary epithelial cells, Mueller cells,
astrocytes, and cells of the trabecular meshwork. The trabecular
meshwork is associated with the passage for fluid drainage out of
the eye. Other cells linked to various ocular-related diseases
include, for example, fibroblasts and vascular endothelial cells.
In that a great deal of retinal damage occurs as a result of edema,
thickening of underlying membranes, and build-up of metabolic
byproducts, preferably the expression vector is administered to an
area of vascular leakage.
[0039] According to the invention, the nucleic acid sequence is
operably linked to regulatory sequences necessary for expression,
i.e., a promoter. A "promoter" is a DNA sequence that directs the
binding of RNA polymerase and thereby promotes RNA synthesis. A
nucleic acid sequence is "operably linked" to a promoter when the
promoter is capable of directing transcription of that nucleic acid
sequence. A promoter can be native or nonnative to the nucleic acid
sequence to which it is operably linked.
[0040] Any promoter (i.e., whether isolated from nature or produced
by recombinant DNA or synthetic techniques) can be used in
connection with the present invention to provide for transcription
of the nucleic acid sequence. The promoter preferably is capable of
directing transcription in a eukaryotic (desirably mammalian) cell.
The functioning of the promoter can be altered by the presence of
one or more enhancers and/or silencers present on the vector.
"Enhancers" are cis-acting elements of DNA that stimulate or
inhibit transcription of adjacent genes. An enhancer that inhibits
transcription also is termed a "silencer." Enhancers differ from
DNA-binding sites for sequence-specific DNA binding proteins found
only in the promoter (which also are termed "promoter elements") in
that enhancers can function in either orientation, and over
distances of up to several kilobase pairs (kb), even from a
position downstream of a transcribed region.
[0041] A comparison of promoter sequences that function in
eukaryotes has revealed conserved sequence elements. Generally,
eukaryotic promoters transcribed by RNA polymerase II are typified
by a "TATA box" centered at approximately position -25, which
appears to be essential for accurately positioning the start of
transcription. The TATA box directs RNA polymerase to begin
transcribing approximately 30 base pairs (bp) downstream in
mammalian systems. The TATA box functions in conjunction with at
least two other upstream sequences located about 40 bp and 110 bp
upstream of the start of transcription. Typically, a so-called
"CCAAT box" serves as one of the two upstream sequences, and the
other often is a GC-rich segment. The CCAAT homology can reside on
different strands of the DNA. The upstream promoter element also
can be a specialized signal such as one of those which have been
described in the art and which appear to characterize a certain
subset of genes.
[0042] To initiate transcription, the TATA box and the upstream
sequences are each recognized by regulatory proteins which bind to
these sites, and activate transcription by enabling RNA polymerase
II to bind the DNA segment and properly initiate transcription.
Whereas base changes outside the TATA box and the upstream
sequences have little effect on levels of transcription, base
changes in either of these elements substantially lower
transcription rates (see, e.g., Myers et al., Science, 229, 242-247
(1985); McKnight et al., Science, 217, 316-324 (1982)). The
position and orientation of these elements relative to one another,
and to the start site, are important for the efficient
transcription of some, but not all, coding sequences. For instance,
some promoters function well in the absence of any TATA box.
Similarly, the necessity of these and other sequences for promoters
recognized by RNA polymerase I or III, or other RNA polymerases,
can differ.
[0043] Accordingly, promoter regions can vary in length and
sequence and can further encompass one or more DNA binding sites
for sequence-specific DNA binding proteins and/or an enhancer or
silencer. Enhancers and/or silencers can similarly be present on a
nucleic acid sequence outside of the promoter per se.
[0044] The present invention preferentially employs a viral
promoter. Suitable viral promoters are known in the art and
include, for instance, cytomegalovirus (CMV) promoters, such as the
CMV immediate-early promoter, promoters derived from human
immunodeficiency virus (HIV), such as the HIV long terminal repeat
promoter, Rous sarcoma virus (RSV) promoters, such as the RSV long
terminal repeat, mouse mammary tumor virus (MMTV) promoters, HSV
promoters, such as the Lap2 promoter or the herpes thymidine kinase
promoter (Wagner et al., Proc. Natl. Acad. Sci., 78, 144-145
(1981)), promoters derived from SV40 or Epstein Barr virus, an
adeno-associated viral promoter, such as the p5 promoter, and the
like. Preferably, the viral promoter is an adenoviral promoter,
such as the Ad2 or Ad5 major late promoter and tripartite leader, a
CMV promoter, or an RSV promoter.
[0045] Many of the above-described promoters are constitutive
promoters. Instead of being a constitutive promoter, the promoter
can be an inducible promoter, i.e., a promoter that is up- and/or
down-regulated in response to appropriate signals. For instance,
the regulatory sequences can comprise a hypoxia driven promoter,
which is active when the ocular neovascularization or age-related
macular degeneration is associated with hypoxia. Other examples of
suitable inducible promoter systems include, but are not limited
to, the IL-8 promoter, the metallothionine inducible promoter
system, the bacterial lacZYA expression system, the tetracycline
expression system, and the T7 polymerase system. Further, promoters
that are selectively activated at different developmental stages
(e.g., globin genes are differentially transcribed from
globin-associated promoters in embryos and adults) can be employed.
The promoter sequence that regulates expression of the nucleic acid
sequence can contain at least one heterologous regulatory sequence
responsive to regulation by an exogenous agent. The regulatory
sequences are preferably responsive to exogenous agents such as,
but not limited to, drugs, hormones, or other gene products. For
example, the regulatory sequences, e.g., promoter, preferably are
responsive to glucocorticoid receptor-hornone complexes, which, in
turn, enhance the level of transcription of a therapeutic peptide
or a therapeutic fragment thereof.
[0046] Preferably, the regulatory sequences comprise a
tissue-specific promoter, i.e., a promoter that is preferentially
activated in a given tissue and results in expression of a gene
product in the tissue where activated. A typically used
tissue-specific promoter is a myocyte-specific promoter. A promoter
exemplary of a myocyte-specific promoter is the myosin light-chain
1A promoter. A tissue specific promoter for use in the present
inventive vector can be chosen by the ordinarily skilled artisan
based upon the target tissue or celltype. Preferred tissue-specific
promoters for use in the present inventive methods are specific to
ocular tissue, such as a rhodopsin promoter. Examples of rhodopsin
promoters include, but are not limited to, a GNAT cone- transducing
alpha-subunit gene promoter or an interphotoreceptor retinoid
binding protein promoter.
[0047] One of ordinary skill in the art will appreciate that each
promoter drives transcription, and, therefore, protein expression,
differently with respect to time and amount of protein produced.
For example, the CMV promoter is characterized as having peak
activity shortly after transduction, i.e., about 24 hours after
transduction, then quickly tapering off On the other hand, the RSV
promoter's activity increases gradually, reaching peak activity
several days after transduction, and maintains a high level of
activity for several weeks. Indeed, sustained expression driven by
an RSV promoter has been observed in all cell types studied,
including, for instance, liver cells, lung cells, spleen cells,
diaphragm cells, skeletal muscle cells, and cardiac muscle cells.
Thus, a promoter can be selected for use in the methods of the
present invention by matching its particular pattern of activity
with the desired pattern and level of expression of at least one
inhibitor of angiogenesis and/or at least one neurotrophic factor.
Alternatively, a hybrid promoter can be constructed which combines
the desirable aspects of multiple promoters. For example, a CMV-RSV
hybrid promoter combining the CMV promoter's initial rush of
activity with the RSV promoter's high maintenance level of activity
would be especially preferred for use in many embodiments of the
present inventive method. It is also possible to select a promoter
with an expression profile that can be manipulated by an
investigator.
[0048] Also preferably, the expression vector comprises a nucleic
acid encoding a cis-acting factor, wherein the cis-acting factor
modulates the expression of the nucleic acid sequence. Preferably,
the cis-acting factor comprises matrix attachment region (MAR)
sequences (e.g., immunoglobulin heavy chain (Jenunwin et al.,
Nature, 385(16), 269 (1997)), apolipoprotein B, or locus control
region (LCR) sequences, among others. MAR sequences have been
characterized as DNA sequences that associate with the nuclear
matrix after a combination of nuclease digestion and extraction
(Bode et al., Science, 255(5041), 195-197 (1992)). MAR sequences
are often associated with enhancer-type regulatory regions and,
when integrated into genomic DNA, MAR sequences augment
transcriptional activity of adjacent nucleotide sequences. It has
been postulated that MAR sequences play a role in controlling the
topological state of chromatin structures, thereby facilitating the
formation of transcriptionally-active complexes. Similarly, it is
believed LCR sequences function to establish and/or maintain
domains permissive for transcription. Many LCR sequences give
tissue specific expression of associated nucleic acid sequences.
Addition of MAR or LCR sequences to the expression vector can
further enhance expression of at least one inhibitor of
angiogenesis and/or at least one neurotrophic factor.
[0049] With respect to promoters, nucleic acid sequences,
selectable markers, and the like, located on an expression vector
according to the present invention, such elements can be present as
part of a cassette, either independently or coupled. In the context
of the present invention, a "cassette" is a particular base
sequence that possesses functions which facilitate subcloning and
recovery of nucleic acid sequences (e.g., one or more restriction
sites) or expression (e.g., polyadenylation or splice sites) of
particular nucleic acid sequences.
[0050] Construction of an exogenous nucleic acid operably linked to
regulatory sequences necessary for expression is well within the
skill of the art (see, for example, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 2d ed. (1989)). With respect to the
expression of nucleic acid sequences according to the present
invention, the ordinary skilled artisan is aware that different
genetic signals and processing events control levels of nucleic
acids and proteins/peptides in a cell, such as, for instance,
transcription, mRNA translation, and post-transcriptional
processing. Transcription of DNA into RNA requires a functional
promoter, as described herein.
[0051] Protein expression is dependent on the level of RNA
transcription that is regulated by DNA signals, and the levels of
DNA template. Similarly, translation of mRNA requires, at the very
least, an AUG initiation codon, which is usually located within 10
to 100 nucleotides of the 5' end of the message. Sequences flanking
the AUG initiator codon have been shown to influence its
recognition by eukaryotic ribosomes, with conformity to a perfect
Kozak consensus sequence resulting in optimal translation (see,
e.g., Kozak, J Molec. Biol., 196, 947-950 (1987)). Also, successful
expression of an exogenous nucleic acid in a cell can require
post-translational modification of a resultant protein. Thus,
production of a protein can be affected by the efficiency with
which DNA (or RNA) is transcribed into mRNA, the efficiency with
which mRNA is translated into protein, and the ability of the cell
to carry out post-translational modification. These are all factors
of which the ordinary skilled artisan is aware and is capable of
manipulating using standard means to achieve the desired end
result.
[0052] Along these lines, to optimize protein production,
preferably the nucleic acid sequence further comprises a
polyadenylation site following the coding region of the nucleic
acid sequence. Also, preferably all the proper transcription
signals (and translation signals, where appropriate) will be
correctly arranged such that the nucleic acid sequence will be
properly expressed in the cells into which it is introduced. If
desired, the nucleic acid sequence also can incorporate splice
sites (i.e., splice acceptor and splice donor sites) to facilitate
mRNA production. Moreover, if the nucleic acid sequence encodes a
protein or peptide, which is a processed or secreted protein or
acts intracellularly, preferably the nucleic acid sequence further
comprises the appropriate sequences for processing, secretion,
intracellular localization, and the like.
[0053] In certain embodiments, it may be advantageous to modulate
expression of the at least one inhibitor of angiogenesis and/or at
least one neurotrophic factor. An especially preferred method of
modulating expression of a nucleic acid sequence comprises addition
of site-specific recombination sites on the expression vector.
Contacting an expression vector comprising site-specific
recombination sites with a recombinase will either up- or
down-regulate transcription of a coding sequence, or simultaneously
up-regulate transcription one coding sequence and down-regulate
transcription of another, through the recombination event. Use of
site-specific recombination to modulate transcription of a nucleic
acid sequence is described in, for example, U.S. Pat. Nos.
5,801,030 and 6,063,627 and International Patent Application WO
97/09439.
[0054] Preferably, the expression vector of the present inventive
method comprises a nucleic acid encoding an inhibitor of
angiogenesis. More preferably, the nucleic acid sequence encodes
multiple inhibitors of angiogenesis. By "inhibitor of angiogenesis"
is meant any factor that prevents or ameliorates
neovascularization. One of ordinary skill in the art will
understand that complete prevention or amelioration of
neovascularization is not required in order to realize a
therapeutic effect. Therefore, the present inventive methods
contemplate both partial and complete prevention and amelioration
of angiogenesis. An inhibitor of angiogenesis includes, for
instance, an anti-angiogenic factor, an anti-sense molecule
specific for an angiogenic factor, a ribozyme, a receptor for an
angiogenic factor, and an antibody that binds a receptor for an
angiogenic factor.
[0055] The anti-angiogenic factors contemplated for use in the
present invention include pigment epithelium-derived factor,
angiostatin, vasculostatin, endostatin, platelet factor 4,
heparinase, interferons (e.g., INF.alpha.), and the like. One of
ordinary skill in the art will appreciate that any anti-angiogenic
factor can be modified or truncated and retain anti-angiogenic
activity. As such, active fragments of anti-angiogenic factors
(i.e., those fragments having biological activity sufficient to
inhibit angiogenesis) are also suitable for use in the present
inventive methods.
[0056] An anti-sense molecule specific for an angiogenic factor
should generally be substantially identical to at least a portion,
preferably at least about 20 continuous nucleotides, of the nucleic
acid encoding the angiogenic factor to be inhibited, but need not
be identical. The anti-sense nucleic acid molecule can be designed
such that the inhibitory effect applies to other proteins within a
family of genes exhibiting homology or substantial homology to the
nucleic acid. The introduced anti-sense nucleic acid molecule also
need not be full-length relative to either the primary
transcription product or fully processed mRNA. Generally, higher
homology can be used to compensate for the use of a shorter
sequence. Furthermore, the anti-sense molecule need not have the
same intron or exon pattern, and homology of non-coding segments
will be equally effective. Antisense phosphorothiotac
oligodeoxynucleotides (PS-ODNs) is exemplary of an anti-sense
molecule specific for an angiogenic factor.
[0057] Ribozymes can be designed that specifically pair with
virtually any target RNA and cleave the phosphodiester backbone at
a specific location, thereby functionally inactivating the target
RNA. In carrying out this cleavage, the ribozyme is not itself
altered and is, thus, capable of recycling and cleaving other
molecules, making it a true enzyme. The inclusion of ribozyme
sequences within antisense RNAs confers RNA-cleaving activity upon
them, thereby increasing the activity of the constructs. The design
and use of target RNA-specific ribozymes is described in Haseloffet
al., Nature, 334, 585-591 (1988). Preferably, the ribozyme
comprises at least about 20 continuous nucleotides complementary to
the target sequence on each side of the active site of the
ribozyme.
[0058] Receptors specific for angiogenic factors inhibit
neovascularization by sequestering growth factors away from
functional receptors capable of promoting a cellular response. For
example, Flt and Flk receptors, as well as VEGF-receptor chimeric
proteins, compete with VEGF receptors on vascular endothelial cells
to inhibit endothelial cell growth (Aiello, PNAS, 92, 10457
(1995)). Also contemplated are growth factor-specific antibodies
and fragments thereof (e.g., Fab, F(ab').sub.2, and Fv) that
neutralize angiogenic factors or bind receptors for angiogenic
factors.
[0059] The present invention also contemplates delivery of a
nucleic acid sequence encoding at least one neurotrophic agent (or
neurotrophic factor) to ocular cells or cells associated with
age-related macular degeneration. Neurotrophic factors are thought
to be responsible for the maturation of developing neurons and for
maintaining adult neurons. Neurotrophic factors are divided into
three subclasses: neuropoietic cytokines; neurotrophins; and the
fibroblast growth factors. Ciliary neurotrophic factor (CNTF) is
exemplary of neuropoietic cytokines. CNTF promotes the survival of
ciliary ganglionic neurons and supports certain neurons that are
NGF-responsive. Neurotrophins include, for example, brain-derived
neurotrophic factor and nerve growth factor, perhaps the best
characterized neurotrophic factor. Other neurotrophic factors
suitable for being encoded by the nucleic acid sequence of the
present inventive methods include, for example, transforming growth
factors, glial cell-line derived neurotrophic factor, neurotrophin
3, neurotrophin 4/5, and interleukin 1-.beta.. Neurotrophic factors
associated with angiogenesis, such as aFGF and bFGF, are less
preferred. The neurotrophic factor of the present inventive method
can also be a neuronotrophic factor, e.g., a factor that enhances
neuronal survival. It has been postulated that neurotrophic factors
can actually reverse degradation of neurons. Such factors,
conceivably, are useful in treating the degeneration of neurons
associated with vision loss. Neurotrophic factors function in both
paracrine and autocrine fashions, making them ideal therapeutic
agents. Preferably, the nucleic acid sequence of the present
invention encodes both an inhibitor of angiogenesis and a
neurotrophic factor. More preferably, the nucleic acid sequence
encodes at least one factor comprising both anti-angiogenic and
neurotrophic properties. Most preferably, the factor comprising
both anti-angiogenic and neurotrophic properties is pigment
epithelium-derived factor (PEDF).
[0060] PEDF, also named early population doubling factor-1 (EPC-1),
is a secreted protein having homology to a family of serine
protease inhibitors named serpins. PEDF is made predominantly by
retinal pigment epithelial cells and is detectable in most tissues
and cell types of the body. PEDF has been observed to induce
differentiation in retinoblastoma cells and enhance survival of
neuronal populations (Chader, Cell Different., 20, 209-216 (1987)).
Factors that enhance neuronal survival under adverse conditions,
such as PEDF, are termed "neuronotrophic," as described herein.
PEDF further has gliastatic activity, or has the ability to inhibit
glial cell growth. As discussed above, PEDF also has antiangiogenic
activity. Anti-angiogenic derivatives of PEDF include SLED
proteins, discussed in WO 99/04806. It has also been postulated
that PEDF is involved with cell senescence (Pignolo et al., J Biol.
Chem., 268(12), 8949-8957 (1998)). PEDF for use in the present
inventive method can be derived from any source, and is further
characterized in U.S. Pat. No. 5,840,686 and International Patent
Applications WO 93/24529 and WO 99/04806.
[0061] In addition to the methods of prophylactically or
therapeutically treating an ocular-related disorder, the present
invention further provides a viral vector comprising a nucleic acid
sequence encoding PEDF or a therapeutic fragment thereof, wherein
the nucleic acid sequence operably linked to regulatory sequences
necessary for expression of PEDF or a therapeutic fragment thereof.
The nucleic acid sequence can be obtained from any source, e.g.,
isolated from nature, synthetically generated, isolated from a
genetically engineered organism, and the like. Appropriate viral
vectors and regulatory sequences are discussed herein. In nature,
PEDF is almost solely generated in human fetus retinal cells. The
poor production of human PEDF from RPE cells and the scarcity of
source tissue of PEDF complicates the use of this potentially
valuable therapeutic factor. The viral vector of the present
invention can be used to create sufficient amounts of recombinant
PEDF or can be used in methods of research or treatment, e.g., the
present inventive method.
[0062] The expression vector, e.g., the adenoviral or the
adeno-associated viral vector, also can comprise a nucleic acid
sequence encoding a therapeutic fragment of at least one inhibitor
of angiogenesis or at least one neurotrophic factor. One of
ordinary skill in the art will appreciate that any inhibitor of
angiogenesis or neurotrophic factor, e.g., PEDF, can be modified or
truncated and retain anti-angiogenic or neurotrophic activity. As
such, therapeutic fragments (i.e., those fragments having
biological activity sufficient to, for example, inhibit
angiogenesis or promote neuron survival) also are suitable for
incorporation into the expression vector. Also suitable for
incorporation into the expression vector are nucleic acid sequences
comprising substitutions, deletions, or additions, but which encode
a functioning inhibitor of angiogenesis or neurotrophic factor or a
therapeutic fragment of any of the foregoing. Likewise, a fusion
protein comprising an anti-angiogenic factor or neurotrophic factor
or a therapeutic fragment thereof and for example, a moiety that
stabilizes peptide conformation, also can be present in the
expression vector. A functioning inhibitor of angiogenesis or a
therapeutic fragment thereof prevents or ameliorates
neovascularization. A functioning neurotrophic factor or a
therapeutic fragment thereof desirably promotes neuronal cell
differentiation, inhibits glial cell proliferation, and/or promotes
neuronal cell survival. One of ordinary skill in the art will
understand that complete prevention or amelioration of
neovascularization is not required in order to realize a
therapeutic effect. Likewise, complete induction of neuron survival
or differentiation is not required in order to realize a benefit.
Therefore, both partial and complete prevention and amelioration of
angiogenesis or promotion of neuron survival is appropriate. The
ordinarily skilled artisan has the ability to determine whether a
modified therapeutic factor or a fragment thereof has neurotrophic
and anti-angiogenic therapeutic activity using, for example,
neuronal cell differentiation and survival assays (see, for
example, U.S. Pat. No. 5,840,686), the mouse ear model of
neovascularization, or the rat hindlimb ischemia model.
[0063] Similarly, one of ordinary skill in the art will appreciate
that the inhibitor of angiogenesis and/or the neurotrophic factor
can be a factor that acts upon a receptor for an anti-angiogenic
factor or a receptor for a neurotrophic factor, thereby resulting
in the desired biological effect. For instance, the expression
vector can comprise a nucleic acid sequence encoding an antibody
that binds and activates the PEDF receptor, which signals a series
of intracellular events responsible for the biological activity of
PEDF. For a discussion of PEDF receptors, see, for example, Alberdi
et al., J Biol. Chem., 274(44), 31605 (1999).
[0064] The present invention also contemplates the use of nucleic
acid sequences encoding chimeric or fusion peptides in the present
inventive method. Through recombinant DNA technology, scientists
have been able to generate fusion proteins that contain the
combined amino acid sequence of two or more proteins. The
ordinarily skilled artisan can fuse the active domains of two or
more factors to generate chimeric peptides with desired activity.
The chimeric peptide can comprise the entire amino acid sequences
of two or more peptides or, alternatively, can be constructed to
comprise portions of two or more peptides (e.g., 10, 20, 50, 75,
100, 400, 500, or more amino acid residues). Desirably, the
chimeric peptide comprises anti-angiogenic and neurotrophic
activity, which can be determined using routine methods.
[0065] As discussed herein, the expression vector of the present
inventive method comprises a nucleic acid sequence that encodes at
least one inhibitor of angiogenesis and/or at least one
neurotrophic factor. Therefore, the nucleic acid sequence can
encode multiple, i.e., two, three, or more, inhibitors of
angiogenesis. Likewise, the nucleic acid sequence can encode
multiple, i.e., two, three, or more, neurotrophic factors. In a
preferred embodiment, the nucleic acid sequence encodes PEDF and
ciliary neurotrophic factor (CNTF). Also preferably, the nucleic
acid sequence encodes at least one inhibitor of angiogenesis and at
least one neurotrophic factor. Multiple inhibitors of angiogenesis
and/or multiple neurotrophic factors can be operably linked to
different promoters. As discussed herein, different promoters have
dissimilar levels and patterns of activity. One of ordinary skill
in the art will appreciate the freedom to dictate the expression of
different coding sequences through the use of multiple promoters.
Alternatively, the multiple coding sequences can be operably linked
to the same promoter to form a polycistronic element. The
polycistronic element is transcribed into a single mRNA molecule
when transduced into the ocular cell. Translation of the mRNA
molecule is initiated at each coding sequence, thereby producing
the multiple, separate peptides simultaneously. The present
invention also contemplates contacting an ocular cell or a cell
associated with age-related macular degeneration with a cocktail of
expression vectors, wherein each expression vector encodes a
different inhibitor of angiogenesis and/or neurotrophic factor. The
cocktail of expression vectors can further comprise different types
of expression vectors, e.g., adenoviral vectors and
adeno-associated viral vectors.
[0066] The methods of the present invention can be part of a
treatment regimen involving other therapeutic modalities. It is
appropriate, therefore, if the ocular-related disorder, namely
ocular neovascularization or age-related macular degeneration, has
been treated, is being treated, or will be treated with any of a
number of ocular therapies, such as drug therapy, photodynamic
therapy, photocoagulation laser therapy, panretinal therapy,
thermotherapy, radiation therapy, or surgery. Preferably, the
surgery is macular translocation, removal of subretinal blood, or
removal of subretinal choroidal neovascular membrane. The
expression vector is preferably administered intraocularly for the
prophylactic or therapeutic treatment of age-related macular
degeneration or persistent or recurrent ocular neovascularization
treated with surgery, laser photocoagulation, and photodynamic
therapies.
[0067] The expression vector is preferably administered as soon as
possible after it has been determined that an animal, such as a
mammal, specifically a human, is at risk for ocular
neovascularization or age-related macular degeneration
(prophylactic treatment) or has begun to develop ocular
neovascularization or age-related macular degeneration (therapeutic
treatment). Treatment will depend, in part, upon the particular
nucleic acid sequence used, the particular inhibitor of
angiogenesis and/or neurotrophic factor expressed from the nucleic
acid sequence, the route of administration, and the cause and
extent, if any, of ocular neovascularization or age-related macular
degeneration realized. For example, systemic administration or
administration to both eyes is preferred in the prophylactic
treatment of macular degeneration because, once one eye is
affected, the other eye is at risk (up to 19% per year).
[0068] The expression vector of the present invention desirably is
administered in a pharmaceutical composition, which comprises a
pharmaceutically acceptable carrier and the expression vector(s).
Any suitable pharmaceutically acceptable carrier can be used within
the context of the present invention, and such carriers are well
known in the art. The choice of carrier will be determined, in
part, by the particular site to which the composition is to be
administered and the particular method used to administer the
composition.
[0069] Suitable formulations include aqueous and non-aqueous
solutions, isotonic sterile solutions, which can contain
anti-oxidants, buffers, bacteriostats, and solutes that render the
formulation isotonic with the blood or intraocular fluid of the
intended recipient, and aqueous and non-aqueous sterile suspensions
that can include suspending agents, solubilizers, thickening
agents, stabilizers, and preservatives. The formulations can be
presented in unit-dose or multi-dose sealed containers, such as
ampules and vials, and can be stored in a freeze-dried
(lyophilized) condition requiring only the addition of the sterile
liquid carrier, for example, water, immediately prior to use.
Extemporaneous solutions and suspensions can be prepared from
sterile powders, granules, and tablets of the kind previously
described. Preferably, the pharmaceutically acceptable carrier is a
buffered saline solution. More preferably, the expression vector
for use in the present inventive methods is administered in a
pharmaceutical composition formulated to protect the expression
vector from damage prior to administration. For example, the
pharmaceutical composition can be formulated to reduce loss of the
expression vector on devices used to prepare, store, or administer
the expression vector, such as glassware, syringes, or needles. The
pharmaceutical composition can be formulated to decrease the light
sensitivity and/or temperature sensitivity of the expression
vector. To this end, the pharmaceutical composition preferably
comprises a pharmaceutically acceptable liquid carrier, such as,
for example, those described above, and a stabilizing agent
selected from the group consisting of polysorbate 80, L-arginine,
polyvinylpyrrolidone, trehalose, and combinations thereof. Use of
such a pharmaceutical composition will extend the shelf life of the
vector, facilitate administration, and increase the efficiency of
the present inventive methods. In this regard, a pharmaceutical
composition also can be formulated to enhance transduction
efficiency.
[0070] In addition, one of ordinary skill in the art will
appreciate that the expression vector, e.g., viral vector, of the
present invention can be present in a composition with other
therapeutic or biologically-active agents. For example, therapeutic
factors useful in the treatment of a particular indication can be
present. For instance, if treating vision loss, hyaluronidase can
be added to a composition to effect the break down of blood and
blood proteins in the vitreous of the eye. Factors that control
inflammation, such as ibuprofen or steroids, can be part of the
composition to reduce swelling and inflammation associated with in
vivo administration of the viral vector and ocular distress. Immune
system suppressors can be administered in combination with the
present inventive method to reduce any immune response to the
vector itself or associated with an ocular disorder.
Anti-angiogenic factors, such as soluble growth factor receptors,
growth factor antagonists, i.e., angiotensin, and the like also can
be part of the composition, as well as additional neurotrophic
factors. Similarly, vitamins and minerals, anti-oxidants, and
micronutrients can be co-administered. Antibiotics, i.e.,
microbicides and fungicides, can be present to reduce the risk of
infection associated with gene transfer procedures and other
disorders.
[0071] One skilled in the art will appreciate that suitable
methods, i.e., invasive and noninvasive methods, of administering
an expression vector whereon the expression vector will contact an
ocular cell are available. Although more than one route can be used
to administer a particular expression vector, a particular route
can provide a more immediate and more effective reaction than
another route. Accordingly, the described routes of administration
are merely exemplary and are in no way limiting.
[0072] The present inventive methods are not dependent on the mode
of administering the expression vector to an animal, preferably a
human, to achieve the desired effect. As such, any route of
administration is appropriate so long as the expression vector
contacts an appropriate ocular cell. The expression vector for use
in the present inventive methods can be appropriately formulated
and administered in the form of an injection, eye lotion, ointment,
implant and the like. The expression vector can be applied, for
example, systemically, topically, subconjunctivally, intraocularly,
retrobulbarly, periocularly, subretinally, or suprachoroidally. In
certain cases, it may be appropriate to administer multiple
applications and employ multiple routes, e.g., subretinal and
intravitreous, to ensure sufficient exposure of ocular cells to the
expression vector. Multiple applications of the expression vector
may also be required to achieve the desired effect.
[0073] Depending on the particular case, it may be desirable to
non-invasively administer the expression vector to a patient. For
instance, if multiple surgeries have been performed, the patient
displays low tolerance to anesthetic, or if other ocular-related
disorders exist, topical administration of the expression vector
may be most appropriate. Topical formulations are well known to
those of skill in the art. Such formulations are suitable in the
context of the present invention for application to the skin. The
use of patches, corneal shields (see, e.g., U.S. Pat. No.
5,185,152), and ophthalmic solutions (see, e.g., U.S. Pat. No.
5,710,182) and ointments, e.g., eye drops, is also within the skill
in the art. The expression vector can also be administered
non-invasively using a needleless injection device, such as the
Biojector 2000 Needle-Free Injection Management System.RTM.
available from Bioject, Inc.
[0074] The expression vector is preferably present in or on a
device that allows controlled or sustained release of the
expression vector, such as an ocular sponge, meshwork, mechanical
reservoir, or mechanical implant. Implants (see, e.g., U.S. Pat.
Nos. 5,443,505, 4,853,224 and 4,997,652), devices (see, e.g., U.S.
Pat. Nos. 5,554,187, 4,863,457, 5,098,443 and 5,725,493), such as
an implantable device, e.g., a mechanical reservoir, an intraocular
device or an extraocular device with an intraocular conduit, or an
implant or a device comprised of a polymeric composition are
particularly useful for ocular administration of the expression
vector. The expression vector of the present inventive methods can
also be administered in the form of sustained-release formulations
(see, e.g., U.S. Pat. No. 5,378,475) comprising, for example,
gelatin, chondroitin sulfate, a polyphosphoester, such as
bis-2-hydroxyethyl-terephthalate (BHET), or a polylactic-glycolic
acid.
[0075] Alternatively, the expression vector can be administered
using invasive procedures, such as, for instance, intravitreal
injection or subretinal injection optionally preceded by a
vitrectomy. Subretinal injections can be administered to different
compartments of the eye, i.e., the anterior chamber. While
intraocular injection is preferred, injectable compositions can
also be administered intramuscularly, intravenously, and
intraperitoneally. Pharmaceutically acceptable carriers for
injectable compositions are well-known to those of ordinary skill
in the art (see Pharmaceutics and Pharmacy Practice,J.B. Lippincott
Co., Philadelphia, Pa., Banker and Chalmers, eds., pages 238-250
(1982), and ASHP Handbook on Injectable Drugs, Toissel, 4.sup.th
ed., pages 622-630 (1986)). Although less preferred, the expression
vector can also be administered in vivo by particle bombardment,
i.e., a gene gun.
[0076] Preferably, the expression vector is administered via an
ophthalmologic instrument for delivery to a specific region of an
eye. Use of a specialized ophthalmologic instrument ensures precise
administration of the expression vector while minimizing damage to
adjacent ocular tissue. Delivery of the expression vector to a
specific region of the eye also limits exposure of unaffected cells
to the inhibitor of angiogenesis and/or neurotrophic factor,
thereby reducing the risk of side effects. A preferred
ophthalmologic instrument is a combination of forceps and
subretinal needle or sharp bent cannula.
[0077] While not particularly preferred, the expression vector can
be administered parenterally. Preferably, any expression vector
parenterally administered to a patient for the prophylactic or
therapeutic treatment of an ocular-related disorder, i.e., ocular
neovascularization or age-related macular degeneration, is
specifically targeted to ocular cells. As discussed herein, an
expression vector can be modified to alter the binding specificity
or recognition of an expression vector for a receptor on a
potential host cell. With respect to adenovirus, such manipulations
can include deletion of regions of the fiber, penton, or hexon,
insertions of various native or non-native ligands into portions of
the coat protein, and the like. One of ordinary skill in the art
will appreciate that parenteral administration can require large
doses or multiple administrations to effectively deliver the
expression vector to the appropriate host cells.
[0078] One of ordinary skill in the art will also appreciate that
dosage and routes of administration can be selected to minimize
loss of expression vector due to a host's immune system. For
example, for contacting ocular cells in vivo, it can be
advantageous to administer to a host a null expression vector
(i.e., an expression vector not comprising the nucleic acid
sequence encoding at least one inhibitor of angiogenesis and/or at
least one neurotrophic factor) prior to performing the present
inventive method. Prior administration of null expression vectors
can serve to create an immunity in the host to the expression
vector, thereby decreasing the amount of vector cleared by the
immune system.
[0079] The dose of expression vector administered to an animal,
particularly a human, in accordance with the present invention
should be sufficient to effect the desired response in the animal
over a reasonable time frame. One skilled in the art will recognize
that dosage will depend upon a variety of factors, including the
age, species, the pathology in question, and condition or disease
state. Dosage also depends on the inhibitor of angiogenesis and/or
neurotrophic factor to be expressed, as well as the amount of
ocular tissue about to be affected or actually affected by the
ocular-related disease. The size of the dose also will be
determined by the route, timing, and frequency of administration as
well as the existence, nature, and extent of any adverse side
effects that might accompany the administration of a particular
expression vector and the desired physiological effect. It will be
appreciated by one of ordinary skill in the art that various
conditions or disease states, in particular, chronic conditions or
disease states, may require prolonged treatment involving multiple
administrations.
[0080] Suitable doses and dosage regimens can be determined by
conventional range-finding techniques known to those of ordinary
skill in the art. Preferably, the about 10.sup.6 viral particles to
about 10.sup.12 viral particles are delivered to the patient. In
other words, a pharmaceutical composition can be administered that
comprises an expression vector concentration of from about 10.sup.6
particles/ml to about 10.sup.12 particles/ml (including all
integers within the range of about 10.sup.6 particles/ml to about
10.sup.12 particles/ml), preferably from about 10.sup.10
particles/ml to about 10.sup.12 particles/ml, and will typically
involve the intraocular administration of from about 0.1 .mu.l to
about 100 .mu.I of such a pharmaceutical composition per eye. When
the expression vector is a plasmid, preferably about 0.5 ng to
about 1000 .mu.g of DNA is administered. More preferably, about 0.1
.mu.g to about 500 .mu.g is administered, even more preferably
about 1 .mu.g to about 100 .mu.g of DNA is administered. Most
preferably, about 50 .mu.g of DNA is administered per eye. Of
course, other routes of administration may require smaller or
larger doses to achieve a therapeutic effect. Any necessary
variations in dosages and routes of administration can be
determined by the ordinarily skilled artisan using routine
techniques known in the art.
[0081] In some embodiments, it is advantageous to administer two or
more (i.e., multiple) doses of the expression vector comprising a
nucleic acid sequence encoding at least one inhibitor of
angiogenesis and/or at least one neurotrophic agent. The present
inventive method provides for multiple applications of the
inhibitor of angiogenesis and/or neurotrophic agent to
prophylactically or therapeutically treat ocular neovascularization
or other ocular-related disorders. For example, at least two
applications of an expression vector comprising an exogenous
nucleic acid, e.g., a nucleic acid sequence encoding at least one
inhibitor of angiogenesis and/or at least one neurotrophic agent,
can be administered to the same eye. Preferably, the multiple doses
are administered while retaining gene expression above background
levels. Also preferably, the ocular cell is contacted with two
applications or more of the expression vector within about 30 days
or more. More preferably, two or more applications are administered
to ocular cells of the same eye within about 90 days or more.
However, three, four, five, six, or more doses can be administered
in any time frame (e.g., 2, 7, 10, 14, 21, 28, 35, 42, 49, 56, 63,
70, 77, 85 or more days between doses) so long as gene expression
occurs and ocular neovascularization is inhibited or
ameliorated.
[0082] It also will be appreciated by one skilled in the art that
an expression vector comprising a nucleic acid sequence encoding at
least one inhibitor of angiogenesis and/or at least one
neurotrophic factor can be introduced ex vivo into cells,
preferably ocular cells, previously removed from a given animal, in
particular a human. Such transduced autologous or homologous host
cells, reintroduced into the animal or human, will express directly
at least one inhibitor of angiogenesis and/or at least one
neurotrophic factor in vivo. One ex vivo therapeutic option
involves the encapsidation of infected ocular cells into a
biocompatible capsule, which can be implanted in the eye or any
other part of the body. One of ordinary skill in the art will
understand that such cells need not be isolated from the patient,
but can instead be isolated from another individual and implanted
into the patient.
[0083] It will be appreciated that the expression vector,
preferably the adenoviral vector, can comprise multiple nucleic
acid sequences encoding the at least one inhibitor of angiogenesis
and/or the at least one neurotrophic factor. For example, the
expression vector can comprise multiple copies of the PEDF coding
sequence, each copy operably linked to a different promoter or to
identical promoters.
[0084] In addition to the above, the nucleic acid sequence encoding
at least one inhibitor of angiogenesis and/or at least one
neurotrophic factor can further comprise one or more other
transgenes. By "transgene" is meant any nucleic acid that can be
expressed in a cell. Desirably, the expression of the transgene is
beneficial, e.g., prophylactically or therapeutically beneficial,
to the ocular cell or eye. If the transgene confers a prophylactic
or therapeutic benefit to the cell, the transgene can exert its
effect at the level of RNA or protein. For example, the transgene
can encode a peptide other than an inhibitor of angiogenesis or
neurotrophic factor that can be employed in the treatment or study
of a disorder, e.g., an ocular-related disorder. Alternatively, the
transgene can encode an antisense molecule, a ribozyme, a protein
that affects splicing or 3' processing (e.g., polyadenylation), or
a protein that affects the level of expression of another gene
within the cell (i.e., where gene expression is broadly considered
to include all steps from initiation of transcription through
production of a process protein), such as by mediating an altered
rate of mRNA accumulation or transport or an alteration in
post-transcriptional regulation. The transgene can encode a
chimeric peptide for combination treatment of an ocular-related
disorder. The transgene can encode a factor that acts upon a
different target molecule than the inhibitor of angiogenesis or the
neurotrophic agent. Indeed, the transgene product can act upon a
different signal transduction pathway, or can act at different
points of the same signal transduction pathway of the inhibitor of
angiogenesis or the neurotrophic factor. Preferably, the
therapeutic substance is a neurotrophic factor, such as CNTF. CNTF
belongs to the neuropoietic cytokines subclass of neurotrophic
factors. CNTF promotes the survival of ciliary ganglionic neurons
and supports certain neurons that are nerve growth factor
(NGF)-responsive.
[0085] Alternatively, one or more additional nucleic acid sequences
(e.g., transgenes) that encode a factor associated with cell
differentiation can be included in the expression vector (e.g.,
viral vector). Preferably, the transgene encodes an
atonal-associated peptide such as Math1 or Hath1 or a biologically
active fragment of either of the foregoing. Math1 is a member of
the mouse basic helix-loop-helix family of transcription factors
and is homologous to the Drosophila gene atonal. Hath1 is the human
counterpart of Math1. Math1 has been shown to be essential for hair
development and can stimulate hair regeneration in the ear.
Combining neurotrophic activity and the hair cell differentiation
properties of an atonal-associated peptide provides a powerful tool
for the treatment and research of, for example, sensory disorders.
Math1 is further characterized in, for example, Bermingham et al.,
Science, 284, 1837-1841 (1999) and Zheng and Gao, Nature
Neuroscience, 3(2), 580-586 (2000).
[0086] The expression vector can comprise a nucleic acid sequence
encoding a vessel maturation factor in addition to at least one
inhibitor of angiogenesis and/or at least one neurotrophic factor.
Many ocular disorders involve leakage of blood products through
vessels, which can cloud vision and induce an immune response
within the layers of the eye. Vessel maturation factors reduce the
amount of vascular leakage and, therefore, are useful in treating,
for example, exudative ocular disorders. Vessel maturation factors
include, but are not limited to, angiopoietins (Ang, e.g., Ang-1
and Ang-2), tumor necrosis factor-alpha (TNF-.alpha.), midkine
(MK), COUP-TFII, and heparin-binding neurotrophic factor (HBNF,
also known as heparin binding growth factor). A nucleotide sequence
encoding an immunosuppressor also can be incorporated into the
expression vector to reduce any inappropriate immune response
within the eye as a result of an ocular-related disorder or the
administration of the expression vector.
[0087] The expression vector can comprise one or more additional
nucleic acid sequences encoding an additional anti-angiogenic
substance. As set forth above, an anti-angiogenic substance is any
biological factor that prevents or ameliorates neovascularization.
One of ordinary skill in the art will understand that the
anti-angiogenic substance can effect partial or complete prevention
and amelioration of angiogenesis to achieve a therapeutic effect.
An anti-angiogenic substance includes, for instance, an
anti-angiogenic factor, an anti-sense molecule specific for an
angiogenic factor, a ribozyme, a receptor for an angiogenic factor,
and an antibody that binds a receptor for an angiogenic factor.
[0088] The transgene can encode a marker protein, such as green
fluorescent protein or luciferase. Such marker proteins are useful
in vector construction and determining vector migration. Marker
proteins also can be used to determine points of injection or
treated ocular tissues in order to efficiently space injections of
the expression vector to provide a widespread area of treatment, if
desired. Alternatively, the transgene can encode a selection
factor, which also is useful in vector construction protocols. If
desired, the transgene can be part of an expression cassette.
[0089] It should be appreciated that any of the nucleic acid
sequences described herein can be altered from their native form to
increase their therapeutic effect. For example, a cytoplasmic form
of a therapeutic nucleic acid can be converted to a secreted form
by incorporating a signal peptide into the encoded gene product.
The at least one inhibitor of angiogenesis and/or at least one
neurotrophic factor can be designed to be taken up by neighboring
cells by fusion of the peptide with VP22. This allows an ocular
cell comprising the therapeutic nucleic acid to have a therapeutic
effect on a number of ocular cells within the mammal.
[0090] The present inventive methods also can involve the
co-administration of other pharmaceutically active compounds. By
"co-administration" is meant administration before, concurrently
with, e.g., in combination with the expression vector in the same
formulation or in separate formulations, or after administration of
the expression vector as described above. For example, factors that
control inflammation, such as ibuprofen or steroids, can be
co-administered to reduce swelling and inflammation associated with
intraocular administration of the expression vector.
Immunosuppressive agents can be co-administered to reduce
inappropriate immune responses related to an ocular disorder or the
practice of the present inventive method. Anti-angiogenic factors,
such as soluble growth factor receptors, growth factor antagonists,
i.e., angiotensin, and the like can also be co-administered, as
well as neurotrophic factors. Similarly, vitamins and minerals,
anti-oxidants, and micronutrients can be co-administered.
Antibiotics, i.e., microbicides and fungicides, can be
co-administered to reduce the risk of infection associated with
ocular procedures and some ocular-related disorders.
[0091] Although the expression vectors of the instant invention are
particularly useful in the study or treatment of ocular disorders,
including ocular disorders comprising a neovascular component and
age-related macular degeneration, it will be appreciated that the
expression vectors can be used to research and/or treat
prophylactically or therapeutically a wide variety of animal
diseases. For example, the viral vector comprising a nucleic acid
sequence encoding PEDF or a therapeutic fragment thereof can be
used in the study or treatment of the nervous system, genitourinary
ailments, cancer, infectious disease, and cardiovascular
abnormalities, as well as miscellaneous other health nuisances. The
viral vector can be used to study or treat, for example, sleep
disorders, ALS (Lou Gehrig's Disease), Alzheimer's Disease,
epilepsy, multiple sclerosis, Parkinson's Disease, peripheral
neuropathies, Schizophrenia, depression, anxiety, spinal cord
injury, traumatic brain injury, or acute, chronic, or inflammatory
pain. The expression vectors of the present invention can be used
to treat genitourinary ailments, which include, for example, benign
prostatic hyperplasia (BPH), impotence, neurogenic bladder, urinary
incontinence, kidney failure, and end stage renal disease. The
expression vectors are useful in treating cancer such as, for
example, cancer of the bladder, brain, breast, colorectal,
esophageal, head & neck, liver/hepatoma, lung, melanoma,
ovarian, pancreatic, prostate, stomach, testicular,
uterine/endometrial, leukemias, and lymphomas. Exemplary infectious
diseases for treatment with the expression vectors include, but are
not limited to, chlamydia, herpes, malaria, human papilloma virus
(HPV), AIDS/HIV, pneumococcal pneumonia, influenza, meningitis,
hepatitis, and tuberculosis. Cardiovascular diseases such as, for
example, neovascular diseases, ischemia, congestive heart failure,
coronary artery disease, arrhythmia, atherosclerosis, increased
LDL/HDL ratios, restenosis after angioplasty or in-stent
restenosis, stroke, sickle cell anemia, and hemophilia, can be
treated or studied, as well as the alleviation of, for example,
obesity, organ transplantation/transplant rejection, osteoporosis,
alopecia, hair loss, arthritis, allergies (such as to ragweed,
pollen, and animal dander), cystic fibrosis, diabetes, and hearing
loss. One of ordinary skill in the art will appreciate that animal
models exist for many of the disease states identified above.
EXAMPLES
[0092] The following examples further illustrate the present
invention but, of course, should not be construed as in any way
limiting its scope.
Example 1
[0093] This example illustrates a preferred method of obtaining
expression of a factor comprising both anti-angiogenic and
neurotrophic activity from an adenoviral vector in in vivo
retina.
[0094] An adenoviral vector deficient in one or more essential gene
functions of the E1, E3, and E4 regions of the adenoviral genome
and comprising a PEDF gene (Ad.PEDF) is preferably constructed as
set forth in WO 99/15686 (McVey et al.). However, the method of the
present invention is not dependent on the method of vector
construction employed and previously described methods of vector
construction are also suitable. Several in vivo models of ocular
neovascularization are available. Neovascularization of the retina
is obtained in, for example, neonatal animals, i.e., neonatal mice,
by exposing the mice to hypoxic conditions shortly after birth.
Several days later, the neonatal mice are exposed to standard
atmospheric conditions, resulting in ischemia-induced
neovascularization of the retina.
[0095] Ad.PEDF is administered to the right eye of at least 12 day
old mice anesthetized with, for example, ketamine or a combination
of ketamine and xylazine via intravitreal injection. Injections are
performed by forming an entrance site in the posterior portion of
the eye and administering approximately 0.1-5.0 .mu.l of
composition comprising Ad.PEDF. In most instances, an injection of
the expression vector will be administered to only one eye, while
the remaining eye serves as a control. The mice are sacrificed at
various time points after administration to determine the extent
and duration of PEDF expression in the retina. The right and left
eyes of each animal are enucleated and either fixed for
histological analysis or prepared for PEDF expression analysis.
Detection of PEDF DNA, PEDF RNA, or PEDF protein can be
accomplished using methods well known in the art, such as PCR and
blotting techniques (see, for example, Sambrook et al., supra).
[0096] To determine the effect of PEDF on neovascularization in
vivo in, for example, a human, indirect ophthalmoscopy of the
retina is ideal. Stereophotographs are useful in detecting
extensive neovascularization, but not appropriate for detecting
subtle lesions.
Example 2
[0097] This example demonstrates a preferred method of obtaining
expression of a factor comprising both anti-angiogenic and
neurotrophic activity from an adenoviral vector in in vivo choroid.
The following example further provides methods for determining the
effect of PEDF on neovascularization.
[0098] An adenoviral vector deficient in one or more essential gene
functions of the E1, E3, and E4 regions of the adenoviral genome
and comprising a PEDF gene (Ad.PEDF) is constructed as set forth in
WO 99/15686 (McVey et al.).
[0099] An in vivo model of choroidal neovascularization can be
obtained by detaching the retina of an eye of, for example, a mouse
or rabbit, and debriding the pigmented epithelia. Choriocapillary
regeneration is monitored in both treated and untreated eyes.
Ad.PEDF is administered prior to perturbing the retinal pigment
epithelial (RPE) to determine the effect of the present inventive
method in preventing choroidal neovascularization. Of course,
Ad.PEDF is administered after perturbing the retina and RPE for
determining the therapeutic effect of the procedure on
neovascularization.
[0100] Choroidal neovascularization can be monitored in vivo using
fundus photography, fluorescein angiography and/or
indocyanine-green angiography, as commonly used in the art. Using
these methods, one of ordinary skill in the art is able to detect
growth of new blood vessels and vascular leakage often associated
with neovascularization. For research purposes, neovascularization
can also be determined by enucleating the eyes and preparing
vascular casts or examining ocular tissue via scanning electron
microscopy.
Example 3
[0101] This example demonstrates the utility of adenoviral vectors
to deliver multiple doses of an exogenous nucleic acid to the
eye.
[0102] Adenoviral vectors comprising the luciferase gene (Ad.L) or
control adenoviral vectors comprising no transgene (Ad.null) were
injected into the intravitreal space of C57BL6 mouse eyes (Day 0).
One day following injection of the adenoviral vectors (Day 1), eyes
infected with Ad.L were enucleated and frozen (1.sup.st
administration). The eyes infected with Ad.nuIl were divided into
three groups. In Group I, Ad.L vectors were injected into the
intravitreal space of the eye at Day 14 (fourteen days following
the initial dose of Ad.null). Group I eyes were enucleated and
frozen the day following the second administration of adenoviral
vectors (Day 15, 2.sup.nd administration). Group II eyes were
injected intravitreally with Ad.null at Day 14, and injected
intravitreally with Ad.L vectors four weeks following the initial
injection with Ad.null (Day 28, 3.sup.rd administration). The eyes
were then enucleated and frozen the day after the third
administration of adenoviral vector. Group III eyes were injected
intravitreally with Ad.null at Day 14 and Day 28, and injected with
Ad.L vectors six weeks following the initial injection with Ad.null
(Day 42, 4.sup.th administration). The eyes were then enucleated
and frozen the day after the fourth administration of adenoviral
vector. Luciferase assays were performed on the eye samples to
determine the efficiency of infection and gene expression
associated with multiple dosing of the vectors.
[0103] Luciferase expression in ocular cells after the 1.sup.st and
2.sup.nd administration of adenoviral vector was substantially
equivalent. In other words, no loss of gene expression was detected
following two administrations of the gene transfer vector. Gene
expression from the 3.sup.rd administration of adenoviral vector
was between 10- and 100-fold reduced compared to gene expression
from the 1.sup.st administration and the 2.sup.nd administration,
but was still above background levels (e.g., as detected in cells
transduced with Ad.null). Gene expression from the 4.sup.th
administration of adenoviral vector was reduced approximately 3- to
10-fold compared to the gene expression observed following the
3.sup.rd administration. However, the level of gene expression
following the 4.sup.th administration was above background
levels.
[0104] This example demonstrates the feasibility of performing
multiple applications of adenoviral vectors to the eye in order to
obtain expression of an exogenous nucleic acid in ocular cells.
Example 4
[0105] This example demonstrates the ability of an expression
vector comprising a nucleic acid sequence encoding a factor
comprising both anti-angiogenic and neurotrophic properties to
inhibit choroidal neovascularization (CNV).
[0106] Replication-deficient (E1-/E3-deficient) adenoviral vectors
(AdPEDF. 10) comprising the coding sequence for PEDF operably
linked to the CMV immediate early promoter were constructed using
standard techniques. A null version of the vector (AdNull. 10),
which did not comprise the PEDF coding sequence, was also
constructed.
[0107] Adult C57BL/6 mice were injected intravitreously with
AdNull. 10 or AdPEDF. 10 using a Harvard pump microinjection
apparatus and pulled glass micropipettes. Each eye was injected
intravitreously with 1 .mu.l of vehicle containing 10.sup.9
particles of virus. Alternatively, each eye was injected
subretinally with 10.sup.8 particles of virus suspended in 1 .mu.l
of vehicle. Five days post-injection, mice were anesthetized with
ketamine hydrochloride (100 mg/kg body weight). Topicamide (1%) was
utilized to dilate the pupils prior to rupture of Bruch's membrane
by diode laser photocoagulation. Rupture of Bruch's membrane is
known to induce neovascularization of the choroid.
[0108] Fourteen days following laser-induced rupture of Bruch's
membrane, choroidal flat mounts (described in Edelman et al.,
Invest. Ophthalmol. Vis. Sci., 41, S834 (2000)) were prepared to
observe the degree of neovascularization of the choroidal membrane.
Briefly, eyes were removed from the subjects and fixed in
phosphate-buffered formalin. The cornea, lens, and retina were
removed from the eyecup, and the eyecup was flatmounted. Flat
mounts were then examined by fluorescence microscopy and images
were digitized using a 3 color CCD video camera (IK-TU40A, Toshiba,
Tokyo, Japan) for computer image analysis.
[0109] Large areas of neovascularization were observed in
uninjected eyes and eyes receiving AdNull. 10. Eyes injected with
AdPEDF. 10 subretinally or intravitreously showed smaller regions
of neovascularization compared to the controls using computerized
image analysis.
[0110] The above results illustrate the ability of the present
inventive method to inhibit ocular neovascularization, namely
choroidal neovascularization (CNV), in a clinically animal relevant
model.
Example 5
[0111] This example demonstrates the ability of an expression
vector comprising a nucleic acid sequence encoding a factor
comprising both anti-angiogenic and neurotrophic properties to
inhibit ischemia-induced retinal neovascularization.
[0112] Replication-deficient adenoviral vectors comprising the
coding sequence for PEDF operably linked to the CMV immediate early
promoter were constructed using standard techniques.
E1-/E3-/E4-deficient vectors encoding PEDF (AdPEDF. 11) and a null
version of the vector (AdNull. 11), which did not comprise the PEDF
coding sequence, were constructed.
[0113] Ischemic retinopathy was produced in adult C57BL/6 mice as
previously described (see, for example, Smith et al., Invest.
Ophthalmol. Vis. Sci., 35, 101 (1994)). Briefly, seven day old mice
(P7) were exposed to an atmosphere of 75 +/-3% oxygen for five
days. At P10, mice were injected intravitreously with 10.sup.9
particles of AdPEDF. 11 or AdNull. 11, returned to oxygen for two
days, then returned to room atmosphere. At P17, the mice were
sacrificed and eyes were rapidly removed and frozen in optimum
cutting temperature embedding compound (OCT; Miles Diagnostics,
Elkhart, Ind.).
[0114] To detect neovascularization, the eyes were sectioned and
histochemically stained with biotinylated griffonia simplicifolia
lectin B4 (GSA, Vector Laboratories, Burlingame, Calif.). Slides
were then incubated in methanol/H.sub.20.sub.2 for 10 minutes at 4
.degree. C., washed with 0.05 M Tris-buffered saline, pH 7.6 (TBS),
and incubated for 30 minutes in 10% normal porcine serum. The
slides were then incubated for two hours with biotinylated GSA,
rinsed with TBS, and incubated with avidin-coupled alkaline
phosphatase (Vector Laboratories) for 45 minutes. After a 10 minute
wash with TBS, the slides were incubated with Histomark Red.
GSA-stained, 10 .mu.m serial sections were examined using an
Axioskop microscope. Images were digitized using a 3 color CCD
video camera (IK-TU40A, Toshiba, Tokyo, Japan) for computer image
analysis.
[0115] Extensive retinal neovascularization was detected in eyes
not injected with any virus. Eyes injected with AdNull. 11 showed
less neovascularization than uninjected eyes, but significantly
more neovascularization of the retina than eyes injected with
AdPEDF. 11. Eyes injected with AdPEDF. 11 comprised the least
amount of neovascularization.
[0116] This example clearly demonstrates the ability of the present
inventive method to inhibit an ocular-related disorder, namely
ischemia-induced retinal neovascularization, in a clinically
relevant animal model.
[0117] All references cited herein are hereby incorporated by
reference to the same extent as if each reference was individually
and specifically indicated to be incorporated by reference and was
set forth in its entirety herein.
[0118] While this invention has been described with an emphasis
upon preferred embodiments, it will be obvious to those of ordinary
skill in the art that variations of the preferred embodiments may
be used and that it is intended that the invention may be practiced
otherwise than as specifically described herein. Accordingly, this
invention includes all modifications encompassed within the spirit
and scope of the invention as defined by the following claims.
* * * * *