Note: Descriptions are shown in the official language in which they were submitted.
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USE OF RECOMBINANT GENE DELIVERY VECTORS FOR TREATING OR
PREVENTING DISEASES OF THE EYE
TECHNICAL FIELD
The present invention relates generally to compositions and methods for
treating diseases of the eye, and more specifically, to the use of various
gene delivery
vectors which direct the expression of selected gene products suitable for
treating or
preventing diseases of the eye.
BACKGROUND OF THE INVENTION
Eye diseases represent a significant health problem in the United States
and world-wide. Presently, over 80 million Americans are affected with
potentially
blinding eye disease, with 1.1 million being legally blind. Twelve million
individuals
suffer from some degree of visual impairment that cannot be corrected. The
total
economic impact of eye disease is also very significant. In 1981, the
estimated
economic impact of visual impairment on the U.S. economy was 14 billion per
year.
By 1995, this impact had grown to an estimated 38.4 billion (National Eye
Institute
NIH).
A wide variety of eye diseases can cause visual impairment, including
for example, macular degeneration, diabetic retinopathies, inherited retinal
degeneration
such as retinitis pigmentosa, glaucoma, retinal detachment or injury and
retinopathies
(whether inherited; induced by surgery, trauma, a toxic compound or agent, or,
photically).
One structure in the eye that can be particularly affected by disease is the
retina. Briefly, the retina, which is found at the back of the eye, is a
specialized light-
sensitive tissue that contains photoreceptor cells (rods and cones) and
neurons
connected to a neural network for the processing of visual information (see
Figure 10).
This information is sent to the brain for decoding into a visual image.
The retina depends on cells of the adjacent retinal pigment epithelium
(RPE) for support of its metabolic functions. Photoreceptors in the retina,
perhaps
because of their huge energy requirements and highly differentiated state, are
sensitive
to a variety of genetic and environmental insults. The retina is thus
susceptible to an
array of diseases that result in visual loss or complete blindness.
Retinitis pigmentosa (RP), which results in the destruction of
photoreceptor cells, the RPE, and choroid typify inherited retinal
degenerations. This
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group of debilitating conditions affects approximately 100,000 people in the
United
States.
A great deal of the progress made in addressing this important clinical
problem has depended on advances in research on photoreceptor cell biology,
molecular
biology, molecular genetics, and biochemistry over the past two decades.
Animal
models of hereditary retinal disease have been vital in helping unravel the
specific
genetic and biochemical defects that underlie abnormalities in human retinal
diseases.
It now seems clear that both genetic and clinical heterogeneity underlie many
hereditary
retinal diseases.
The leading cause of visual loss in the elderly is Age-related Macular
Degeneration (AMD). The social and economic impact of this disease in the
United
States is increasing. The macula is a structure near the center of the retina
that contains
the fovea. This specialized portion of the retina is responsible for the high-
resolution
vision that permits activities such as reading. The loss of central vision in
AMD is
devastating. Degenerative changes to the macula (maculopathy) can occur at
almost
any time in life but are much more prevalent with advancing age. With growth
in the
aged population, AMD will become a more prevalent cause of blindness than both
diabetic retinopathy and glaucoma combined. Laser treatment has been shown to
reduce the risk of extensive macular scarring from the "wet" or neovascular
form of the
disease. The effects of this treatment are short-lived, however, due to
recurrent
choroidal neovascularization. Thus, there are presently no effective
treatments in
clinical use for the vast majority of AMD patients.
Other diseases of the eye, such as glaucoma, are also major public health
problems in the United States. In particular, blindness from glaucoma is
believed to
impose significant costs annually on the U.S. Government in Social Security
benefits,
lost tax revenues, and healthcare expenditures.
Briefly, glaucoma is not a uniform disease but rather a heterogeneous
group of disorders that share a distinct type of optic nerve damage that leads
to loss of
visual function. The disease is manifest as a progressive optic neuropathy
that, if left
untreated, leads to blindness. It is estimated that as many as 3 million
Americans have
glaucoma and, of these, as many as 120,000 are blind as a result. Furthermore,
it is the
number one cause of blindness in African-Americans. Its most prevalent form,
primary
open-angle glaucoma, can be insidious. This form usually begins in midlife and
progresses slowly but relentlessly. If detected early, disease progression can
frequently
be arrested or slowed with medical and surgical treatment.
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Glaucoma can involve several tissues in the front and back of the eye.
Commonly, but not always, glaucoma begins with a defect in the front of the
eye. Fluid
in the anterior portion of the eye, the aqueous humor, forms a circulatory
system that
brings nutrients and supplies to various tissues. Aqueous humor enters the
anterior
chamber via the ciliary body epithelium (inflow), flows through the anterior
segment
bathing the lens, iris, and cornea, and then leaves the eye via specialized
tissues known
as the trabecular meshwork and Schlemm's canal to flow into the venous system.
Intraocular pressure is maintained vis-a-vis a balance between fluid secretion
and fluid
outflow. Almost all glaucomas are associated with defects that interfere with
aqueous
humor outflow and, hence, lead to a rise in intraocular pressure. The
consequence of
this impairment in outflow and elevation in intraocular pressure is that optic
nerve
function is compromised. The result is a distinctive optic nerve atrophy,
which
clinically is characterized by excavation and cupping of the optic nerve,
indicative of
loss of optic nerve axons.
Primary open-angle glaucoma is, by convention, characterized by
relatively high intraocular pressures believed to arise from a blockage of the
outflow
drainage channel or trabecular meshwork in the front of the eye. However,
another
form of primary open-angle glaucoma, normal-tension glaucoma, is characterized
by a
severe optic neuropathy in the absence of abnormally high intraocular
pressure.
Patients with normal-tension glaucoma have pressures within the normal range,
albeit
often in the high normal range. Both these forms of primary open-angle
glaucoma are
considered to be late-onset diseases in that, clinically, the disease first
presents itself
around midlife or later. However, among African-Americans, she disease may
begin
earlier than middle age. In contrast, juvenile open-angle glaucoma is a
primary
glaucoma that affects children and young adults. Clinically, this rare form of
glaucoma
is distinguished from primary open-angle glaucoma not only by its earlier
onset but also
by the very high intraocular pressure associated with this disease. Angle-
closure
glaucoma is a mechanical form of the disease caused by contact of the iris
with the
trabecular meshwork, resulting in blockage of the drainage channels that allow
fluid to
escape from the eye. This form of glaucoma can be treated effectively in the
very early
stages with laser surgery. Congenital and other developmental glaucomas in
children
tend to be severe and can be very challenging to treat successfully. Secondary
glaucomas result from other ocular diseases that impair the outflow of aqueous
humor
from the eye and include pigmentary glaucoma, pseudoexfoliative glaucoma, and
glaucomas resulting from trauma and inflammatory diseases. Blockage of the
outflow
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channels by new blood vessels (neovascular glaucoma) can occur in people with
retinal
vascular disease, particularly diabetic retinopathy.
Primary open-angle glaucoma can be insidious. It usually begins in
midlife and progresses slowly but relentlessly. If detected, disease
progression can
frequently be arrested or slowed with medical and surgical treatment. However,
without treatment, the disease can result in absolute irreversible blindness.
In many
cases, even when patients have received adequate treatment (e.g., drugs to
lower
intraocular pressure), optic nerve degeneration and loss of vision continues
relentlessly.
The present invention provides compositions and methods for treating
and preventing a number of diseases of the eye, including for example, retinal
diseases
and degenerations such as RP and AMD, as well as other diseases such as
neovascular
disease. The present invention also provides other, related advantages.
SUMMARY OF THE INVENTION
Briefly stated, the present invention provides compositions and methods
for treating, preventing, or, inhibiting diseases of the eye. Within one
aspect of the
present invention, methods are provided for treating or preventing diseases of
the eye
comprising the step of intraocularly administering a gene delivery vector
which directs
the expression of one or more neurotrophic factors, or, anti-angiogenic
factors, such that
the disease of the eye is treated or prevented. Within related aspects of the
present
invention, gene delivery vectors are provided which direct the expression of
one or
more neurotrophic factors such as FGF, as well as gene therapy vectors which
direct the
expression of one or more anti-angiogenic factors. Within certain embodiments
of the
invention, a viral promoter (e.g., CMV) or an inducible promoter (e.g., tet)
is utilized to
drive the expression of the neurotrophic factor.
Representative examples of gene delivery vectors suitable for use within
the present invention may be generated from viruses such as retroviruses
(e.g., FIV or
HIV), herpesviruses, adenoviruses, adeno-associated viruses, and alphaviruses,
or from
non-viral vectors.
Utilizing the methods and gene delivery vectors provided herein a wide
variety of diseases of the eye may be readily treated or prevented, including
for
example, glaucoma, macular degeneration, diabetic retinopathies, inherited
retinal
degeneration such as retinitis pigmentosa, retinal detachment or injury and
retinopathies
(whether inherited, induced by surgery, trauma, a toxic compound or agent, or,
photically). Similarly, a wide variety of neurotrophic factors may be utilized
(either
alone or in combination) within the context of the present invention,
including for
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example, NGF, BDNF, CNTF, NT-3, NT-4, FGF-2, FGF-5, FGF-18, FGF-20 and FGF-
21.
Within certain embodiments of the invention, it is preferred that the gene
delivery vector be utilized to deliver and express an anti-angiogenic factor
for the
5 treatment, prevention, or, inhibition of diabetic retinopathy, wet ARMD, and
other
neovascular diseases of the eye (e.g., ROP). Within other embodiments it is
desirable
that the gene delivery vector be utilized to deliver and express a
neurotrophic growth
factor to treat, prevent, or, inhibit diseases of the eye, such as, for
example, glaucoma,
retinitis pigmentosa, and dry ARMD. Within yet other embodiments, it may be
desirable to utilize either a gene delivery vector which expresses both an
anti-
angiogenic molecule and a neurotrophic growth factor, or two separate vectors
which
independently express such factors, in the treatment, prevention, or
inhibition of an eye
disease (e.g., for diabetic retinopathy).
Within further embodiments of the invention, the above-mentioned
methods utilizing gene delivery vectors may be administered along with other
methods
or therapeutic regimens, including for example, photodynamic therapy (e.g.,
for wet
ARMD), laser photocoagulation (e.g., for diabetic retinopathy and wet ARMD),
and
intraocular pressure reducing drugs (e.g., for glaucoma).
Also provided by the present invention are isolated nucleic acid
molecules comprising the sequence of Figure 2, vectors which contain, and/or
express
this sequence, and host cells which contain such vectors.
Within further aspects of the present invention gene delivery vectors are
provided which direct the expression of a neurotrophic factor, or, an anti-
angiogenic
factor. As noted above, representative examples of neurotrophic factors
include NGF,
BDNF, CNTF, NT-3, NT-4, FGF-2, FGF-5, FGF-18, FGF-20 and FGF-21.
Representative examples of anti-angiogenic factors include soluble Flt-1,
soluble Tie-2
receptor, and PEDF. Representative examples of suitable gene delivery vectors
include
adenovirus, retroviruses (e.g., HIV or FIV-based vectors), alphaviruses, AAV
vectors,
and naked DNA vectors.
These and other aspects of the present invention will become evident
upon reference to the following detailed description and attached drawings. In
addition,
various references are set forth herein which describe in more detail certain
procedures
or compositions (e.g., plasmids, etc.), and are therefore incorporated by
reference in
their entirety.
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BRIEF DESCRIPTION OF THE DRAWINGS
Figure 1 is a schematic illustration of pKm201bFGF-2.
Figure 2 is the nucleic acid sequence of pKm201bFGF-2 (SEQ LD.
No. ~.
Figure 3 is a schematic illustration of pD 10-CMV-FGF-5.
Figure 4 is a western analysis of FGF-5 rAAV infected 293 cells.
Figure 5 is a schematic illustration of pD 10-CMV-FGF-5 (sig-).
Figure 6 is a western analysis of pDlO-CMV-FGF-5 (sig-) transfected
293 cells.
Figure 7 is a schematic illustration of pD 10-CMV-FGF-18.
Figure 8 western analysis of 293 cells transfected pDlO-CMV-FGF-18.
Figures 9A and 9B are photographs which show that bluo-gal staining is
visible across 40% of a retina transfected with AAV-CMV-lacZ. All
photoreceptors
appear to express lacZ at the injection site, except at the edge where
individual cells are
visible.
Figure 10 is a schematic illustration which shows the retina within the
eye, and the organization of cells within the retina.
Figures 11A and 11B are photographs of wild-type and degenerated
S334ter rat retinas. S334ter is a rat model for retinitis pigmentosa.
Figures 12A, 12B and 12C are photographs of degenerated S334ter,
FGF-2 injected S334ter and PBS injected S334ter rat retinas. As can be seen in
these
figures, FGF-2 injected into the S334ter rat retina substantially slows the
progression of
disease.
Figure 13 is a graph which plots Outer Nuclear Layer (ONL) thickness
for FGF-2 subretinally injected, PBS subretinally injected, and an uninfected
control.
Figure 14 is a bar graph which plots ONL thickness at p60.
Figures 15A, 15B and 15C are photographs of FGF-2 expressing cells
stained with an anti-FGF-2 antibody.
Figures 16A and 16B are photographs which show gene delivery to cells
in the ganglion cell layer following intraocular injection of recombinant rAAV-
CMV-
lacZ. a) superior quadrant of a retinal flat-mount processed for Bluo-Gal
staining to
visualize AAV-infected neurons. Notice the large number of axons converging at
the
optic nerve head (asterisk). b) Retinal radial section showing the AAV-
mediated LacZ
gene product in cells of the ganglion cell layer. A large number of these
cells can be
identified as RGCs because of the intense LacZ staining in axons projecting to
the optic
nerve head (asterisk). RPE: retinal pigment epithelium, PS: photoreceptor
segments,
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ONL: outer nuclear layer, OPL: outer plexiform layer, INL: inner nuclear
layer, IPL:
inner plexiform layer, GCL: ganglion cell layer. Scale bars: a) 0.5 mm, b) 50
Vim.
Figure 17 is a graph which shows the time-course of AAV-mediated
transgene expression in the ganglion cell layer of the adult rat retina. A
recombinant
AAV vector (rAAV-CMV-IacZ) was injected into the vitreous chamber of adult
rats
and 2, 4 and 8 weeks later, Lac-Z positive neurons in the ganglion cell layer
(GCL)
were counted in retinal flat-mounts. The values are the mean of 3-4 retinas
per time
point ~ standard deviation (p<0.001).
Figures 18A and 18B are photographs which show the localization of the
AAV-mediated LacZ gene product in retrogradely labeled RGCs. a) Retinal radial
section showing LacZ immunopositive RGCs transduced with AAV (excitation 520
560, barner 580, emission 580); b) Same section examined under a different
fluorescent
filter (excitation 355-425, barrier 460, emission 470) to visualize RGCs
backlabeled
with Fluorogold from the superior colliculus. Notice that the vast majority of
LacZ
immunopositive neurons are also labeled with Fluorogold. An exception, a LacZ
positive cell that is not Fluorogold labeled, is shown (arrow), and could
represent a
displaced amacrine cell or RGC that did not incorporate the retrograde tracer.
INL:
inner nuclear layer, IPL: inner plexiform layer; GCL: ganglion cell layer.
Scale bar: 10
Vim.
Figure 19 is a graph which quantifies Fluorogold- and LacZ-positive
cells in the ganglion cell layer following intravitreal injection of rAAV-CMV-
lacZ. The
number of Fluorogold-positive cells (FG+) was compared to the number of cells
that
expressed both the Fluorogold and LacZ markers (FG+, LacZ+) 4,ud the number of
cells
expressing only the reporter gene (LacZ+). Values represent the mean of 4-5
retinal
radial sections per animal (n=4) + standard deviation (S.D.) (p<0.001).
Figure 20 is a photograph which shows the localization of the heparan
sulfate (HS) proteoglycan receptor, the cellular receptor for AAV, in the
intact adult rat
retina. Retinal cryosection immunostained with a polyclonal antibody against
the
heparan sulfate (HepSS-1; diluted 1:200) followed by biotinylated anti-rabbit
Fab
fragment, avidin-biotin-peroxidase reagent (ABC Elite Vector Labs, Burlingame,
CA).
The section was reacted in a solution containing 0.05% diaminobenzidine
tetrahydrochloride (DAB) and 0.06% hydrogen peroxide in PB (pH 7.4) for 5 min.
Notice the strong labeling in RGCs in the ganglion cell layer (GCL). RPE:
retinal
pigment epithelium, PS: photoreceptor segments, ONL: outer nuclear layer, OPL:
outer
plexiform layer, INL: inner nuclear layer, IPL: inner plexiform layer. Scale
bar: 50 Vim.
Figure 21 is a schematic illustration of pDlO-VEGFuc.
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Figure 22 is the nucleotide sequence of pDlO-VEGFuc.
Figure 23 is a bar graph which shows pDlO-VEGFuc rAAV virus
infection of 293 cells.
Figure 24 is a three dimensional bar-graph which shows VEGF secretion
by hfRPE after infection with VEGF AAV.
Figure 25 is a three dimensional bar-graph which shows VEGF secretion
by hfRPE after infection with VEGF AV
Figure 26 is a three dimensional bar-graph which shows resistance of
hfRPE after infection with VEGF AV.
Figure 27 is a schematic illustration of pDlO-sFlt-1.
Figure 28 is the nucleotide sequence of pDlO-sFlt-1.
Figure 29 is the nucleotide sequence of FGF-20.
Figure 30 is the nucleotide sequence of FGF-21.
Figure 31 is a schematic illustration of pDlOK-FGF-2Sc.
Figure 32 is the nucleotide sequence of pDlOK-FGF-2Sc.
Figure 33 is a graph which compares ONL thickness (um) after injection
of various vectors into the eye.
DETAILED DESCRIPTION OF THE INVENTION
DEFINITIONS
Prior to setting forth the invention, it may be helpful to an understanding
thereof to first set forth definitions of certain terms that will be used
hereinafter.
"Gene delivery vector" refers to a construct which is capable of
delivering, and, within preferred embodiments expressing, one or more genes)
or
sequences) of interest in a host cell. Representative examples of such vectors
include
viral vectors, nucleic acid expression vectors, naked DNA, and certain
eukaryotic cells
(e.g., producer cells).
"Recombinant adeno-associated virus vector" or "rAAV vector" refers to
a gene delivery vector based upon an adeno-associated virus. The rAAV vectors,
should contain 5' and 3' adeno-associated virus inverted terminal repeats
(ITRs), and a
transgene or gene of interest operatively linked to sequences which regulate
its
expression in a target cell. Within certain embodiments, the transgene may be
operably
linked to a heterologous promoter (such as CMV), or, an inducible promoter
such as
(tet). In addition, the rAAV vector may have a polyadenylation sequence.
"Neurotrophic Factor" or "NT" refers to proteins which are responsible
for the development and maintenance of the nervous system. Representative
examples
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of neurotrophic factors include NGF, BDNF, CNTF, NT-3, NT-4, and Fibroblast
Growth Factors.
"Fibroblast Growth Factor" or "FGF" refers to a family of related
proteins, the first of which was isolated from the pituitary gland (see
Gospodarowicz,
D., Nature, 249:123-127, 1974). From this original FGF (designated basic FGF)
a
family of related proteins, protein muteins, and protein analogs have been
identified
(see, e.g., U.S. Patent Nos. 4,444,760, 5,155,214, 5,371,206, 5,464,774,
5,464,943,
5,604,293, 5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and
5,872,226), all
of which are generally referred to as Fibroblast Growth Factors within the
context of the
present invention.
"Anti-an_gio~enic Factor" refers to a factor or molecule which is able to
inhibit the proliferation of vascular growth. A variety of assays may be
utilized to
assess the anti-angiogenic activity of a given molecule, including for
example, the assay
provided in Example 15, which measures HUVEC (human umbilical vein endothelial
cell) proliferation. Representative examples of anti-angiogenic factors
include for
example, Angiostatin, 1,25-Di-hydroxy-vitamn D3, Endostatin, IGF-1 receptor
antagonists, Interferons alpha, beta and gamma, Interferon gamma-inducible
protein IP-
10, Interleukin 1 alpha and beta, Interleukin 12, 2-Methoxyestradiol, PEDF,
Platelet
factor 4, Prolactin (l6kd fragment), Protamin, Retinoic acid, Thrombospondin-1
and 2,
Tissue inhibitor of metalloproteinase-1 and -2, Transforming growth factor
beta,
soluble Tie-2 receptor, soluble Tie-2 receptor, soluble Flt-1 and Tumor
necrosis factor -
alpha.
"Diseases of the Eve" refers to a broad class of diseases wherein the
functioning of the eye is affected due to damage or degeneration of the
photoreceptors;
ganglia or optic nerve; or neovascularization. Representative examples of such
diseases
include macular degeneration, diabetic retinopathies, inherited retinal
degeneration such
as retinitis pigmentosa, glaucoma, retinal detachment or injury and
retinopathies
(whether inherited, induced by surgery, trauma, a toxic compound or agent, or,
photically).
As noted above, the present invention provides compositions and
methods for treating, preventing, or, inhibiting diseases of the eye,
comprising the
general step of administering intraocularly a recombinant adeno-associated
viral vector
which directs the expression of one or more neurotrophic factors, such that
the disease
of the eye is treated or prevented. In order to further an understanding of
the invention,
a more detailed discussion is provided below regarding (A) gene delivery
vectors; (B)
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Neurotrophic Factors; (C) Anti-angiogenic factors; and (D) methods of
administering
the rAAVs in the treatment or prevention of diseases of the eye.
A. Gene Deliver~Vectors
1. Construction of retroviral gene delivery vectors
5 Within one aspect of the present invention, retroviral gene delivery
vectors are provided which are constructed to carry or express a selected
genes) or
sequences) of interest. Briefly, retroviral gene delivery vectors of the
present invention
may be readily constructed from a wide variety of retroviruses, including for
example,
B, C, and D type retroviruses as well as spumaviruses and lentiviruses (see
RNA Tumor
10 Viruses, Second Edition, Cold Spring Harbor Laboratory, 1985). Such
retroviruses may
be readily obtained from depositories or collections such as the American Type
Culture
Collection ("ATCC"; Rockville, Maryland), or isolated from known sources using
commonly available techniques.
Any of the above retroviruses may be readily utilized in order to
assemble or construct retroviral gene delivery vectors given the disclosure
provided
herein, and standard recombinant techniques (e.g., Sambrook et al., Molecular
Cloning.
A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, 1989;
Kunkel,
PNAS 82:488, 1985). In addition, within certain embodiments of the invention,
portions of the retroviral gene delivery vectors may be derived from different
retroviruses. For example, within one embodiment of the invention, retrovirus
LTRs
may be derived from a Murine Sarcoma Virus, a tRNA binding site from a Rous
Sarcoma Virus, a packaging signal from a Murine Leukemia Virus, and an origin
of
second strand synthesis from an Avian Leukosis Virus.
Within one aspect of the present invention, retrovector constructs are
provided comprising a 5' LTR, a tRNA binding site, a packaging signal, one or
more
heterologous sequences, an origin of second strand DNA synthesis and a 3' LTR,
wherein the vector construct lacks gaglpol or env coding sequences.
Other retroviral gene delivery vectors may likewise be utilized within the
context of the present invention, including for example EP 0,415,731; WO
90/07936;
WO 91/0285, WO 9403622; WO 9325698; WO 9325234; U.S. Patent No. 5,219,740;
WO 9311230; WO 9310218; Vile and Hart, Cancer Res. 53:3860-3864, 1993; Vile
and
Hart, Cancer Res. 53:962-967, 1993; Ram et al., Cancer Res. 53:83-88, 1993;
Takamiya et al., J. Neurosci. Res. 33:493-503, 1992; Baba et al., J.
Neurosurg. 79:729-
735, 1993 (U.S. Patent No. 4,777,127, GB 2,200,651, EP 0,345,242 and
W091/02805).
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Packaging cell lines suitable for use with the above described retrovector
constructs may be readily prepared (see U.S. Serial No. 08/240,030, filed May
9, 1994;
see also U.S. Serial No. 07/800,921, filed November 27, 1991), and utilized to
create
producer cell lines (also termed vector cell lines or "VCLs") for the
production of
recombinant vector particles.
2. Recombinant Adeno-Associated Virus Vectors
As noted above, a variety of rAAV vectors may be utilized to direct the
expression of one or more desired neurotrophic factors. Briefly, the rAAV
should be
comprised of, in order, a 5' adeno-associated virus inverted terminal repeat,
a transgene
or gene of interest operatively linked to a sequence which regulates its
expression in a
target cell, and a 3' adeno-associated virus inverted terminal repeat. In
addition, the
rAAV vector may preferably have a polyadenylation sequence.
Generally, rAAV vectors should have one copy of the AAV ITR at each
end of the transgene or gene of interest, in order to allow replication,
packaging, and
efficient integration into cell chromosomes. The ITR consists of nucleotides 1
to 145 at
the 5' end of the AAV DNA genome, and nucleotides 4681 to 4536 (i.e., the same
sequence) at the 3' end of the AAV DNA genome. Preferably, the rAAV vector may
also include at least 10 nucleotides following the end of the ITR (i.e., a
portion of the
"D region").
Within preferred embodiments of the invention, the transgene sequence
will be of about 2 to S kb in length (or alternatively, the transgene may
additionally
contain a "stuffer" or "filler" sequence to bring the total sir:; of the
nucleic acid
sequence between the two ITRs to between 2 and 5 kb). Alternatively, the
transgene
may be composed of same heterologous sequence several times (e.g., two nucleic
acid
molecules which encode FGF-2 separated by a ribosome readthrough, or
alternatively,
by an Internal Ribosome Entry Site or "IRES"), or several different
heterologous
sequences (e.g., FGF-2 and FGF-5, separated by a ribosome readthrough or an
IRES).
Recombinant AVV vectors of the present invention may be generated
from a variety of adeno-associated viruses, including for example, serotypes 1
through
6. For example, ITRs from any AAV serotype are expected to have similar
structures
and functions with regard to replication, integration, excision and
transcriptional
mechanisms.
Within certain embodiments of the invention, expression of the
transgene may be accomplished by a separate promoter (e.g., a viral promoter).
Representative examples of suitable promoters in this regard include a CMV
promoter,
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RSV promoter, SV40 promoter, or MoMLV promoter. Other promoters that may
similarly be utilized within the context of the present invention include cell
or tissue
specific promoters (e.g., a rod, cone, or ganglia derived promoter), or
inducible
promoters. Representative examples of suitable inducible promoters include
tetracycline-response promoters ("Tet", see, e.g., Gossen and Bujard, Proc.
Natl. Acad.
Sci. USA. 89:5547-5551, 1992; Gossen et al., Science 268, 1766-1769, 1995;
Baron et
al., Nucl. Acids Res. 25:2723-2729, 1997; Blau and Rossi, Proc. Natl. Acad.
Sci. USA.
96:797-799, 1999; Bohl et al., Blood 92:1512-1517, 1998; and Haberman et al.,
Gene
Therapy 5:1604-1611, 1998), the ecdysone system (see e.g., No et al., Proc.
Natl. Acad.
Sci. USA. 93:3346-3351, 1996), and other regulated promoters or promoter
systems
(see, e.g., Rivera et al., Nat. Med. 2:1028-1032, 1996;).
The rAAV vector may also contain additional sequences, for example
from an adenovirus, which assist in effecting a desired function for the
vector. Such
sequences include, for example, those which assist in packaging the rAAV
vector in
adenovirus-associated virus particles.
Packaging cell lines suitable for producing adeno-associated viral vectors
may be readily accomplished given readily available techniques (see e.g., U.S.
Patent
No. 5,872,005).
Particularly preferred methods for constructing and packaging rAAV
vectors are described in more detail below in Examples 1, 2, 3, and 4.
3. Al~havirus delivery vectors
The present invention also provides a variety of Alphavirus vectors
which may function as gene delivery vectors. For example, the Sindbis virus is
the
prototype member of the alphavirus genus of the togavirus family. The
unsegmented
genomic RNA (495 RNA) of Sindbis virus is approximately 11,703 nucleotides in
length, contains a 5' cap and a 3' poly-adenylated tail, and displays positive
polarity.
Infectious enveloped Sindbis virus is produced by assembly of the viral
nucleocapsid
proteins onto the viral genomic RNA in the cytoplasm and budding through the
cell
membrane embedded with viral encoded glycoproteins. Entry of virus into cells
is by
endocytosis through clatharin coated pits, fusion of the viral membrane with
the
endosome, release of the nucleocapsid, and uncoating of the viral genome.
During viral
replication the genomic 49S RNA serves as template for synthesis of the
complementary negative strand. This negative strand in turn serves as template
for
genomic RNA and an internally initiated 265 subgenomic RNA. The Sindbis viral
nonstructural proteins are translated from the genomic RNA while structural
proteins
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13
are translated from the subgenomic 265 RNA. All viral genes are expressed as a
polyprotein and processed into individual proteins by post translational
proteolytic
cleavage. The packaging sequence resides within the nonstructural coding
region,
therefore only the genomic 495 RNA is packaged into virions.
Several different Sindbis vector systems may be constructed and utilized
within the present invention. Representative examples of such systems include
those
described within U.S. Patent Nos. 5,091,309 and 5,217,879, and PCT Publication
No.
WO 95/07994.
4. Other viral gene delivery vectors
In addition to retroviral vectors and alphavirus vectors, numerous other
viral vectors systems may also be utilized as a gene delivery vector.
Representative
examples of such gene delivery vectors include viruses such as pox viruses,
such as
canary pox virus or vaccinia virus (Fisher-Hoch et al., PNAS 86:317-321, 1989;
Flexner
et al., Ann. N Y. Acad. Sci. 569:86-103, 1989; Flexner et al., Vaccine 8:17-
21, 1990;
U.S. Patent Nos. 4,603,112, 4,769,330 and 5,017,487; WO 89/01973); SV40
(Mulligan
et al., Nature 277:108-114, 1979); influenza virus (Luytjes et al., Cell
59:1107-1113,
1989; McMicheal et al., N. Eng. J. Med. 309:13-17, 1983; and Yap et al.,
Nature
273:238-239, 1978); herpes (Kit, Adv. Exp. Med. Biol. 215:219-236, 1989; U.S.
Patent
No. 5,288,641); HIV (Poznansky, J. Virol. 65:532-536, 1991); measles (EP 0
440,219);
Semliki Forest Virus, and coronavirus, as well as other viral systems (e.g.,
EP
0,440,219; WO 92/06693; U.S. Patent No. 5,166,057). In addition, viral
carriers may
be homologous, non-pathogenic(defective), replication competent virus (e.g.,
Overbaugh et al., Science 239:906-910,1988), and nevertheless induce cellular
immune
responses, including CTL.
5. Non-viral gene delivery vectors
In addition to the above viral-based vectors, numerous non-viral gene
delivery vectors may likewise be utilized within the context of the present
invention.
Representative examples of such gene delivery vectors include direct delivery
of nucleic
acid expression vectors, naked DNA alone (WO 90/11092), polycation condensed
DNA
linked or unlinked to killed adenovirus (Curiel et al., Hum. Gene Ther. 3:147-
154,
1992), DNA ligand linked to a ligand with or without one of the high affinity
pairs
described above (Wu et al., J. of Biol. Chem 264:16985-16987, 1989), nucleic
acid
containing liposomes (e.g., WO 95/24929 and WO 95/12387) and certain
eukaryotic
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14
cells (e.g., producer cells - see U.S. Serial No. 08/240,030, filed May 9,
1994, and U.S.
Serial No. 07/800,921).
B. Neurotrophic Factors
As noted above, the term neurotrophic factor refers to proteins which are
responsible for the development and maintenance of the nervous system.
Representative examples of neurotrophic factors include NGF, BDNF, CNTF, NT-3,
NT-4, and Fibroblast Growth Factors.
Fibroblast Growth Factor refers to a family of related proteins, the first
of which was isolated from the pituitary gland (see Gospodarowicz, D., Nature,
249:123-127, 1974). From this original FGF (designated basic FGF) a family of
related
proteins, protein muteins, and protein analogs have been identified (see,
e.g., U.S.
Patent Nos. 4,444,760, 5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293,
5,731,170, 5,750,365, 5,851,990, 5,852,177, 5,859,208, and 5,872,226; see
generally
Baird and Gospodarowicz, D. Ann N Y. Acad. Sci. 638:1, 1991. The first two
members
of the family to be identified were acidic fibroblast growth factor (aFGF/FGF-
1) and
basic fibroblast growth factor (bFGF/FGF-2). Additional members of the FGF
family
include: i-nt-2lFGF-3, (Smith et al., EMBO J. 7: 1013, 1988); FGF-4 (Delli-
Bovi et al.,
Cell 50: 729, 1987); FGF-6 (Marics et al., Oncogene 4: 335 (1989);
keratinocyte growth
factor/FGF-7, (Finch et al., Science 245: 752, 1989); FGF-8 (Tanaka et al.,
Proc. Natl.
Acad Sci. USA 89: 8928, 1992); and FGF-9 (Miyamoto et al., Mol. Cell Biol. 13:
4251,
1993).
FGF-5 was originally isolated as an oncogene. See Goldfarb et al. US
Patent Nos. 5,155,217 and 5,238,916, Zhan et al. "Human Oncogene Detected by a
Defined Medium Culture Assay" (Oncogene 1:369-376, 1987), Zhan et al. "The
Human
FGF-5 Oncogene Encodes a Novel Protein Related to Fibroblastic Growth Factors"
(Molecular and Cellular Biology 8:3487-3495, 1988), and Bates et al.
"Biosynthesis of
Human Fibroblast Growth Factor 5": (Molecular and Cellular Biology 11:1840-
1845,
1991).
Other FGFs include those disclosed in U.S. Patent Nos. 4,444,760,
5,155,214, 5,371,206, 5,464,774, 5,464,943, 5,604,293, 5,731,170, 5,750,365,
5,851,990, 5,852,177, 5,859,208, and 5,872,226. 5,852,177, and 5,872,226, as
well as
FGF-20 (U.S. Provisional Application No. 60/161,162) and FGF-21 (U.S.
Provisional
Application No. 60/166,540).
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WO 00/54813 PCT/US00/07062
C. Anti-Angiogenic Factors
A wide variety of anti-angiogenic factors may also be expressed from the
gene delivery vectors of the present invention, including for example,
Angiostatin
(O'Reilly et al., Cell 79:315-328, 1994; O'Reilly et al., Nat. Med. 2:689-92,
1996; Sim
5 et al., Cancer Res. 57:1329-34, 1997), 1,25-Di-hydroxy-vitamn D3 (Shibuya et
al.,
Oncogene 5:519-24, 1990; Oikawa et al., Eur. J. Pharmacol. 178:247-50, 1990;
and
182:616, 1990), Endostatin (O'Reilly et al., Cell 88:277-85, 1997),
Interferons alpha and
beta (Sidky et al., Cancer Res. 47:5155-61, 1987; Singh et al., Proc. Natl.
Acad. Sci.
USA 92:4562-6, 1995), Interferon gamma (Friesel et al., J. Cell. Biol. 104:689-
96,
10 1987), IGF-1 receptor antagonists, Interferon gamma-inducible protein IP-10
(Arenberg
et al., J. Exp. Med. 1996;184:981-92; Strieter et al., J. Leukoc. Biol.
1995;57:752-62;
Angiolillo et al., J. Exp. Med. 182:155-62, 1995), Interleukin lalpha and beta
(Cozzolino et al., Proc. Natl. Acad. Sci. USA 87:6487-91, 1990), Interleukin
12 (Kerbel
and Hawley, J. Natl Cancer Inst. 87:557-9, 1995; Majewski et al., J. Invest.
Dermatol
15 106:1114-8, 1996; Voest et al., .l. Natl Cancer Inst. 87:581-6, 1995), 2-
Methoxyestradiol (Fotsis et al., Nature 368:237-9, 1994), PEDF, Platelet
factor 4
(Taylor and Folkman, Nature 297:307-12, 1982; Gengrinovitch et al., J. Biol.
Chem
270:15059-65, 1995), Prolactin (l6kd fragment) (Clapp et al., Endocrinology
133:1292-
9, 1993; Ferrara, Endocrinology 129:896-900, 1991), Protamin, Retinoic acid
(Lingen
et al., Lab. Invest 74:476-83, 1996), Thrombospondin-1 and 2 (Lawler, Blood,
67:1197-
209, 1986; Raugi and Lovett, Am. J. Pathol 129:364-72, 1987; Volpert et al.,
Biochem.
Biophys. Res. Commun 217:326-32, 1995), Tissue inhibitor of metalloproteinase-
1 and
-2 (Moses and Langer, J. Cell Biochem 47:230-5, 1991; Ray :xnd Stetler-
Stevenson,
Eur. Respir. J. 7:2062-72, 1994), Transforming growth factor beta
(RayChaudhury, J.
Cell. Biochem 47:224-9, 1991; Roberts et al., Proc. Natl Acad. Sci USA 83:4167-
71,
1986), and Tumor necrosis factor - alpha (Frater-Schroeder et al., Proc. Natl.
Acad. Sci.
USA 84:5277-81, 1987; Leibovich et al., Nature 329:630-2, 1987).
Other anti-angiogenic factors that can be utilized within the context of
the present invention include VEGF antagonists such as soluble Flt-1 (Kendall
and
Thomas, PNAS 90: 10705, 1993), and Ang-1 antagonists such as soluble Tie-2
receptor
(Thurston et al., Science 286:2511, 1999; see also, generally Aiello et al.,
PNAS
92:10457, 1995; Robinson et al., PNAS 93:4851, 1996; Seo et al., Am. J.
Pathol.
154:1743, 1999).
The ability of a given molecule to be "anti-angiogenic" can be readily
assessed utilizing a variety of assays, including for example, the HUVEC assay
provided in Example 15.
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D. Method for Treating and/or Preventing Diseases of the Eye, and
Pharmaceutical
Compositions
As noted above, the present invention provides methods which generally
comprise the step of intraocularly administering a gene delivery vector which
directs the
expression of one or more neurotrophic factor to the eye, or an anti-
angiogenic factor to
the eye in order to treat, prevent, or inhibit the progression of an eye
disease. As
utilized herein, it should be understood that the terms "treated, prevented,
or, inhibited"
refers to the alteration of a disease course or progress in a statistically
significant
manner. Determination of whether a disease course has been altered may be
readily
assessed in a variety of model systems, discussed in more detail below, which
analyze
the ability of a gene delivery vector to delay, prevent or rescue
photoreceptors, as well
as other retinal cells, from cell death.
1. Diseases of the eye
A wide variety of diseases of the eye may be treated given the teachings
provided herein. For example, within one embodiment of the invention gene
delivery
vectors are administered to a patient intraocularly in order to treat or
prevent macular
degeneration. Briefly, the leading cause of visual loss in the elderly is
macular
degeneration (MD), which has an increasingly important social and economic
impact in
the United States. As the size of the elderly population increases in this
country, age
related macular degeneration (AMD) will become a more prevalent cause of
blindness
than both diabetic retinopathy and glaucoma combined. Although laser treatment
has
been shown to reduce the risk of extensive macular scarnng from the "wet" or
neovascular form of the disease, there are currently no effective treatments
for the vast
majority of patients with MD.
Within another embodiment, gene delivery vectors can be administered
to a patient intraocularly in order to treat or prevent an inherited retinal
degeneration.
One of the most common inherited retinal degenerations is retinitis pigmentosa
(RP),
which results in the destruction of photoreceptor cells, and the RPE. Other
inherited
conditions include Bardet-Biedl syndrome (autosomal recessive); Congenital
amaurosis
(autosomal recessive); Cone or cone-rod dystrophy (autosomal dominant and X-
linked
forms); Congenital stationary night blindness (autosomal dominant, autosomal
recessive
and X-linked forms); Macular degeneration (autosomal dominant and autosomal
recessive forms); Optic atrophy, autosomal dominant and X-linked forms);
Retinitis
pigmentosa (autosomal dominant, autosomal recessive and X-linked forms);
Syndromic
or systemic retinopathy (autosomal dominant, autosomal recessive and X-linked
forms);
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and Usher syndrome (autosomal recessive). This group of debilitating
conditions
affects approximately 100,000 people in the United States alone.
As noted above, within other aspects of the invention, gene delivery
vectors which direct the expression of a neurotrophic growth factor can be
administered
to a patient intraocularly in order to treat or prevent glaucoma. Briefly,
glaucoma is not
a uniform disease but rather a heterogeneous group of disorders that share a
distinct
type of optic nerve damage that leads to loss of visual function. The disease
is manifest
as a progressive optic neuropathy that, if left untreated leads to blindness.
It is
estimated that as many as 3 million Americans have glaucoma and, of these, as
many as
120,000 are blind as a result. Furthermore, it is the number one cause of
blindness in
African-Americans. Its most prevalent form, primary open-angle glaucoma, can
be
insidious. This form usually begins in midlife and progresses slowly but
relentlessly. If
detected early, disease progression can frequently be arrested or slowed with
medical
and surgical treatment. Representative factors that may be expressed from the
vectors
of the present invention to treat glaucoma include neurotrophic growth factors
such as
FGF-2, 5, 18, 20, and, 21.
Within yet other embodiments gene delivery vectors can be administered
to a patient intraocularly in order to treat or prevent injuries to the
retina, including
retinal detachment, photic retinopathies, surgery-induced retinopathies, toxic
retinopathies, retinopathies due to trauma or penetrating lesions of the eye.
As noted above, the present invention also provides methods of treating,
preventing, or, inhibiting neovascular disease of the eye, comprising the step
of
administering to a patient a gene delivery vector which directs the expression
of an anti-
angiogenic factor. Representative examples of neovascular diseases include
diabetic
retinopathy, ARMD (wet form), and retinopathy of preinaturity. Briefly,
choroidal
neovascularization is a hallmark of exudative or wet Age-related Macular
Degeneration
(AMD), the leading cause of blindness in the elderly population. Retinal
neovascularization occurs in diseases such as diabetic retinapathy and
retinopathy of
prematurity (ROP), the most common cause of blindness in the young.
Particularly preferred vectors for the treatment, prevention, or, inhibition
of neovascular diseases of the eye direct the expression of an anti-angiogenic
factor
such as, for example, soluble tie-2 receptor or soluble Flt-1.
2. Animal Models
In order to assess the ability of a selected gene therapy vector to be
effective for treating diseases of the eye which involve neovascularization, a
novel
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18
model for neovascularization (either choroidal or subretinal) can be generated
by
subretinal injection of a recombinant virus (rAV or rAAV) containing an
angiogenic
transgene such as VEGF and/or angiopoietin. The extent and duration of
neovascularization induced by rAAV and rAV vectors containing an angiogenic
transgene such as VEGF can be determined using fundus photography, fluorescein
angiography and histochemistry.
To assess the ability of anti-angiogenic molecules to prevent
neovascularization in the model described above, a D10- sFlt-1 rAAV (or other
gene
delivery vector which directs the expression of an anti-angiogenic factor) is
intraocularly injected, either by subretinal or intravitreal routes of
injection. Generally,
subretinal injection of the gene delivery vector may be utilized to achieve
delivery to
both the choroidal and inner retinal vasculature. Intravitreal injection can
be utilized to
infect Muller cells and retinal ganglion cells (RGCs), which deliver anti-
angiogenic
protein to the retinal vasculature. Muller cells span the retina and would
secrete the
therapeutic protein into the subretinal space.
Such injections may be accomplished either prior to, simultaneous with,
or subsequent to administration of an angiogenic factor or gene delivery
vector which
expresses an angiogenic factor. After an appropriate time interval, inhibition
of
neovascularization can be determined using fundus photography, fluorescein
angiography and/or histochemistry.
While there are many animal models of retinal neovascularization such
as oxygen-induced ischemic retinopathy (Aiello et al., PNAS 93: 4881, 1996.)
and the
VEGF transgenic mouse (Okamoto et al., Am. J. Pathol. 151: 281, 1997), there
are
fewer models of choroidal neovascularization (e.g., laser photocoagulation as
described
by Murata et al., IOVS 39: 2474, 1998). Subretinal neovascularization from the
retinal
rather than choroidal blood supply is also observed in VEGF transgenic animals
(Okamoto et al., Am. J. Pathol. 151: 281, 1997). Hypoxic stimulation of VEGF
expression is known to correlate with neovascularization in human ocular
disease.
The pathologic hallmark of glaucomatous optic neuropathy is the
selective death of retinal ganglion cells (RGCs) (Nickells, R.W., J. Glaucoma
5:345
356. 1996; Levin, L.A. and Louhab, A., Arch. Ophthalmol. 114:488-491, 1996.;
Kerrigan, L.A., Zack, D.J., Quigley, H.A., Smith, S.D. and Pease, M.E., Arch.
Ophthalmol. 115:1031-1035, 1997). Recent studies indicate that RGCs die with
characteristics of apoptosis after injury to the axons of adult RGCs such as
axotomy of
the optic nerve (ON), and in glaucoma and anterior ischemic optic neuropathy
in
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19
humans (Nickells, 1996). Thus, damage to the optic nerve by axotomy is used by
many
researchers as a model for selective apoptotic cell death of adult RGCs.
The loss of RGCs caused by ON transection in adult mammals varies
from 50% to more than 90% depending on the techniques used to identify RGCs,
the
proximity of the lesion to the eye, and the age and species of the animal. For
example,
in a study in adult rats, in which retrogradely transported tracers were used
to
distinguish RGCs from displaced amacrine cells (Villegas-Perez, M.P., Vidal-
Sanz, M.,
Bray, G.M. and Aguayo, A.J., J. Neurosci. 8:265-280, 1988). ON transection
near the
eye (0.5-1 mm) leads to the loss of more than 90% of the RGCs by 2 weeks. In
contrast, in adult animals in which the ON was cut nearly 10 mm from the eye,
54% of
RGCs survived by 3 months (Richardson, P.M., Issa, V.M.K. and Shemie, S., J.
Neurocytol. 11:949-966, 1982.).
Briefly, the posterior pole of the left eye and the origin of the optic nerve
(ON) are exposed through a superior temporal intraorbital approach. A
longitudinal
excision of the ON dural sheath is performed. The ON is then gently separated
from the
dorsal aspect of this sheath and completely transected within the orbit,
within 1 mm of
the optic disc. Care is taken to avoid damage to the ophthalmic artery, which
is located
on the inferomedial dural sheath of the ON.
RGC survival and death following gene delivery can also be examined
using two alternative models of ON injury: 1 ) ON crush; and (2) increased
intraocular
pressure. In the first model the ON is exposed, and then clamped at a distance
of about
one millimeter from the posterior pole using a pair of calibrated forceps as
previously
described (Li et al., Invest. Ophthalmol. Vis. Sci. 40:1004, 1999). In the
second model,
chronic moderately elevated intraocular pressure can be produced unilaterally
by
cauterization of three episcleral vessels as described by Neufeld et al. in
PNAS 17:9944,
1999).
A variety of animal models can be utilized for photoreceptor
degeneration, including the RCS rat model, P23H transgenic rat model, the rd
mouse,
and the S334ter transgenic rat model.
Briefly, in the S334ter transgenic rat model, a mutation occurs resulting
in the truncation of the C-terminal 15 amino acid residues of rhodopsin (a
seven-
transmembrane protein found in photoreceptor outer segments, which acts as a
photopigment). The S334ter mutation is similar to rhodopsin mutations found in
a
subset of patients with retinitis pigmentosa (RP). RP is a heterogeneous group
of
inherited retinal disorders in which individuals experience varying rates of
vision loss
due to photoreceptor degeneration. IN many RP patients, photoreceptor cell
death
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progresses to blindness. Transgenic S334ter rats are born with normal number
of
photoreceptors. The mutant rhodopsin gene begins expression at postnatal day 5
in the
rat, and photoreceptor cell death begins at postnatal day 10-15. In transgenic
line
S334ter-3 , approximately 70% of the outer nuclear layer has degenerated by
day 60 in
5 the absence of any therapeutic intervention. The retinal degeneration in
this model is
consistent from animal to animal and follows a predictable and reproducible
rate. This
provides an assay for therapeutic effect by morphological examination of the
thickness
of the photoreceptor nuclear layer and comparison of the treated eye to the
untreated
(contralateral) eye in the same individual animal.
10 S334ter rats are utilized as a model for RP as follows. Briefly, S334ter
transgenic rats are euthanized by overdose of carbon dioxide inhalation and
immediately perfused intracardially with a mixture of mixed aldehydes (2%
formaldehyde and 2.5 % glutaraldehyde). Eyes are removed and embedded in epoxy
resin, and 1 ~m thick histological sections are made along the vertical
meridian. Tissue
15 sections are aligned so that the ROS and Miiller cell processes crossing
the inner
plexiform layer are continuous throughout the plane of section to assure that
the
sections are not oblique, and the thickness of the ONL and lengths of RIS and
ROS are
measured. These retinal thickness measurements are plotted and establish the
baseline
retinal degeneration rates for the animal model. The assessment of retinal
thickness is
20 as follows: briefly, 54 measurements of each layer or structure were made
at set points
around the entire retinal section. These data were either averaged to provide
a single
value for the retina, or plotted as a distribution of thickness or length
across the retina.
The greatest 3 contiguous values for ONL thickness in each retina is also
compared in
order to determine if any region of retina (e.g., nearest the injection site)
showed
proportionally greater rescue; although most of these values were slightly
greater than
the overall mean of all 54 values, they were no different from control values
than the
overall mean. Thus, the overall mean was used in the data cited, since it was
based on a
much larger number of measurements.
One particularly preferred line of transgenic rats, TgN(s334ter) line 4
(abbreviated s334ter 4) can be utilized for in vivo experiments. Briefly, in
this rat
model expression of the mutated opsin transgene begins at postnatal day PS in
these
rats, leading to a gradual death of photoreceptor cells. These rats develop an
anatomically normal retina up to P15, with the exception of a slightly
increased number
of pyknotic photoreceptor nuclei in the outer nuclear layer (ONL) than in non
transgenic control rats. In this animal model , the rate of photoreceptor cell
death is
approximately linear until P60, resulting in loss of 40-60% of the
photoreceptors.
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After P60, the rate of cell loss decreases, until by one year the retinas have
less than a
single row of photoreceptor nuclei remaining.
3. Methods of Administration
Gene delivery vectors of the present invention may be administered
intraocularly to a variety of locations depending on the type of disease to be
treated,
prevented, or, inhibited, and the extent of disease. Examples of suitable
locations
include the retina (e.g., for retinal diseases), the vitreous, or other
locations in or
adjacent to the eye.
Briefly, the human retina is organized in a fairly exact mosaic. In the
fovea, the mosaic is a hexagonal packing of cones. Outside the fovea, the rods
break up
the close hexagonal packing of the cones but still allow an organized
architecture with
cones rather evenly spaced surrounded by rings of rods. Thus in terms of
densities of
the different photoreceptor populations in the human retina, it is clear that
the cone
density is highest in the foveal pit and falls rapidly outside the fovea to a
fairly even
density into the peripheral retina (see Osterberg, G. (1935) Topography of the
layer of
rods and cones in the human retina. Acta Ophthal. (suppl.) 6, 1-103; see also
Curcio, C.
A., Sloan, K. R., Packer, O., Hendrickson, A. E. and Kalina, R. E. (1987)
Distribution
of cones in human and monkey retina: individual variability and radial
asymmetry.
Science 236, 579-582).
Access to desired portions of the retina, or to other parts of the eye may
be readily accomplished by one of skill in the art (see, generally Medical and
Surgical
Retina: Advances, Controversies, and Management, Hilel Lewis, Stephen J. Ryan,
Eds.,
medical illustrator, Timothy C. Hengst. St. Louis: Mosby, c1994. xix, 534; see
also
Retina, Stephen J. Ryan, editor in chief,. 2nd ed., St. Louis, Mo.: Mosby,
c1994. 3 v.
(xxix, 2559 p.).
The amount of the specific viral vector applied to the retina is uniformly
quite small as the eye is a relatively contained structure and the agent is
injected directly
into it. The amount of vector that needs to be injected is determined by the
intraocular
location of the chosen cells targeted for treatment. The cell type to be
transduced will
be determined by the particular disease entity that is to be treated.
For example, a single 20-microliter volume (of 10'3 physical particle
titer/ml rAAV) may be used in a subretinal injection to treat the macula and
fovea. A
larger injection of 50 to 100 microliters may be used to deliver the rAAV to a
substantial fraction of the retinal area, perhaps to the entire retina
depending upon the
extent of lateral spread of the particles.
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A 100-ul injection will provide several million active rAAV particles
into the subretinal space. This calculation is based upon a titer of 10'3
physical particles
per milliliter. Of this titer, it is estimated that 1/1000 to 1/10,000 of the
AAV particles
are infectious. The retinal anatomy constrains the injection volume possible
in the
S subretinal space (SRS). Assuming an injection maximum of 100 microliters,
this would
provide an infectious titer of 108 to 109 rAAV in the SRS. This would have the
potential
of infecting all of the 150 x 10 6 photoreceptors in the entire human retina
with a single
inj ection.
Smaller injection volumes focally applied to the fovea or macula may
adequately transfect the entire region affected by the disease in the case of
macular
degeneration or other regional retinopathies.
Gene delivery vectors can alternately be delivered to the eye by
intraocular injection into the vitreous. In this application, the primary
target cells to be
transduced are the retinal ganglion cells, which are the retinal cells
primarily affected in
glaucoma. In this application, the injection volume of the gene delivery
vector could be
substantially larger, as the volume is not constrained by the anatomy of the
subretinal
space. Acceptable dosages in this instance can range from 25 ul to 1000 ul.
4. As_ says
A wide variety of assays may be utilized in order to determine
appropriate dosages for administration, or to assess the ability of a gene
delivery vector
to treat or prevent a particular disease. Certain of these assays are
discussed in more
detail below.
a. Electroretino _~raphic analXsis
Electroretinographic analysis can be utilized to assess the effect of gene
delivery administration into the retina. Briefly, rats are dark adapted
overnight and then
in dim red light, then anesthetized with intramuscular injections of xylazine
(13 mg/kg)
and ketamine (87 mg/kg). Full-field scotopic ERGS are elicited with 10-sec
flashes of
white light and responses were recorded using a UTAS-E 2000 Visual
Electrodiagnostic
System (LKC Technologies, Inc., Gaithersburg, MD). The corneas of the rats are
the
anesthetized with a drop of 0.5% proparacaine hydrochloride, and the pupils
dilated
with 1% atropine and 2.5% phenylephrine hydrochloride. Small contact lenses
with
gold wire loops are placed on both corneas with a drop of 2.5% methylcellulose
to
maintain corneal hydration. A silver wire reference electrode is placed
subcutaneously
between the eyes and a ground electrode is placed subcutaneously in the hind
leg.
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23
Stimuli are presented at intensities of -1.1, 0.9 and 1.9 log cd m-2 at 10-
second, 30-
second and 1-minute intervals, respectively. Responses are amplified at a gain
of 4,000,
filtered between 0.3 to 500 Hz and digitized at a rate of 2,000 Hz on 2
channels. Three
responses are averaged at each intensity. The a-waves are measured from the
baseline
to the peak in the cornea-negative direction, and b-waves are measured from
the cornea-
negative peak to the major cornea-positive peak. For quantitative comparison
of
differences between the two eyes of rats, the values from all the stimulus
intensities are
averaged for a given animal.
b. Retinal tissue anal,
As described in more detail above and below, retinal tissue analysis can
also be utilized to assess the effect of gene delivery administration into the
retina.
5. Pharmaceutical Compositions
Gene delivery vectors may be prepared as a pharmaceutical product
suitable for direct administration. Within preferred embodiments, the vector
should be
admixed with a pharmaceutically acceptable carrier for intraocular
administration.
Examples of suitable carriers are saline or phosphate buffered saline.
DEPOSIT INFORMATION
The following material was deposited with the American Type Culture
Collection:
Name Deposit Date Accession No.
PKm201bFGF-2 3/11/99 #207160
PD 10-Kan-FGF-2-Sc
The above material was deposited by Chiron Corporation with the
American Type Culture Collection (ATCC), 10801 University Blvd., Mantissas, VA
20110-2209, telephone 703-365-2700. This deposit will be maintained under the
terms
of the Budapest Treaty on the International Recognition of the Deposit of
Microorganisms for purposes of Patent Procedure. The deposit will be
maintained for a
period of 30 years following issuance of this patent, or for the enforceable
life of the
patent, whichever is greater. Upon issuance of the patent, the deposits will
be available
to the public from the ATCC without restriction.
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24
This deposit is provided merely as a convenience to those of skill in the
art, and is not an admission that a deposit is required under 35 U.S.C. ~ 112.
The
nucleic acid sequence of this deposit, as well as the amino acid sequence of
the
polypeptide encoded thereby, are incorporated herein by reference and should
be
referred to in the event of an error in the sequence described herein. A
license may be
required to make, use, or sell the deposited materials, and no such license is
granted
hereby.
The following examples are offered by way of illustration, and not by
way of limitation.
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EXAMPLES
EXAMPLE 1
CONSTRUCTION OF A RAAV VECTOR EXPRESSING FGF-2
5 pKm201 CMV is an AAV cloning vector in which an expression cassette,
consisting of a CMV immediate early promoter/enhancer and a bovine growth
hormone
(BGH) polyadenylation site, is flanked by inverted terminal repeat (ITR)
sequences
from AAV-2. Briefly, pKm201CMV was derived from pKm201, a modified AAV
vector plasmid in which the ampicillin resistance gene of pEMBL-AAV-ITR (see
10 Srivastava, (1989) Proc. Natl. Acad. Sci. USA 86:8078-8082) had been
replaced with
the gene for kanamycin resistance. The expression cassette from pCMVlink, a
derivative of pCMV6c (see Chapman, Nucleic Acids Res. 19:193-198 (1991)) in
which
the BGH poly A site has been substituted for the SV40 terminator, was inserted
between the ITRs of pKm201 to generate pKm201 CMV.
15 pKm201bFGF-2 was constructed by cloning the following, in order, into
the multiple cloning site of pKm201CMV: the encephalomyocarditis virus (EMCV)
internal ribosome entry site (IRES), the bovine FGF-2 cDNA, and the human
growth
hormone polyadenylation sequence. The cDNA for FGF-2 has two mutations that
change amino acid 121 from serine to threonine and amino acid 137 from proline
to
20 serine. The DNA sequence of pKm201bFGF-2 is shown in Figure 2 and the
plasmid
has been deposited with the American Type Culture Collection (ATCC).
rAAV vector particles were produced by a triple transfection protocol
(Nucleic Acids Res. 24:596-601, 1996; J. Exp. Med. 179:1867-1875, 1994).
Briefly,
human embryonic kidney 293 cells, grown to 50% confluence in a 10 layer
Nunclon
25 cell factory (Nalge Nunc, Int., Naperville, IL), were co-transfected with
400 ~g of
helper plasmid pKSrep/cap (Hum. Gene Ther. 9:477-485, 1998) 400 pg of vector
plasmid, and 800 p,g of adenovirus plasmid pBHGlO (Microbix Biosystems, Inc.,
Toronto, Ontario) using the calcium phosphate co-precipitation method. Forty-
eight
hours after co-transfection, media was replaced with IMDM + 10% FBS containing
adenovirus type 5 d1312 at a multiplicity of infection (MOI) of 2. Seventy-two
hours
after infection cells were harvested and resuspended in HEPES buffer (2.5 ml
per dish)
and lysed by three cycles of freezing and thawing. Cell debris was removed by
centrifugation at 12,OOOX g for 20 min. Packaged rAAV was purified from
adenovirus
by two rounds of cesium chloride equilibrium density gradient centrifugation.
Residual
adenovirus contamination was inactivated by heating at 56°C for 45 min.
Though three
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26
plasmids were used in the production of rAAV vector in this example, it is
possible to
combine the rAAV vector construct and the AAV helper gene construct on one
plasmid.
This would allow rAAV to be produced by transfecting 293 cells with two
plasmids.
Alternatively, one could add the adenovirus helper genes to this plasmid to
make a
single plasmid containing all that is required to make rAAV particles.
To estimate total number of rAAV particles, the virus stock was treated
with DNAse I, and encapsidated DNA was extracted with phenol-chloroform, and
precipitated with ethanol. DNA dot blot analysis against a known standard was
used to
determine titer (Blood 76:1997-2000, 1990).
To assay for adenovirus contamination, 293 cells were infected with 10
~l of purified rAAV stock and followed for any signs of cytopathic effect. All
stocks
were negative for adenovirus contamination (level of detection greater than or
equal to
100 PFU/ml).
To assay for wild type AAV, 293 cells were co-infected with serial
dilutions of rAAV stocks and adenovirus d1312 at a MOI of 2. Three days later
the cells
were harvested, lysed by three cycles of freezing/thawing, and centrifuged to
remove
cell debris. The supernatant was heat inactivated (56°C for 10 min) and
fresh plates of
293 cells (6 x 106) were infected in the presence of adenovirus d1312 at a MOI
of 2.
Forty-eight hours after infection, low molecular-weight DNA was isolated (J.
Mol. Biol.
26:365-369, 1967) subjected to agarose gel electrophoresis, and transferred to
a nylon
membrane. The membrane was hybridized with a biotinylated oligonucleotide
probe
specific for the AAV capsid region. The wild type AAV titer was defined as the
highest
dilution of rAAV vector stock demonstrating a positive hybridization signal.
The
rAAV preparations contained less than 1 wild type AAV genome per 109 rAAV
genomes.
EXAMPLE 2
INFECTION OF CELLS WITH RAAV-CMV-FGF-2 RESULTS IN THE EXPRESSION OF FGF-2
293 cells were plated the day before infection at 5 x 10' cells/well in 6-
well plates and were infected with rAAV-CMV-bFGF-2 virus, prepared as
described
above in Example l, at different multiplicities of infection (MOI) with and
without
etoposide (3~M). Etoposide is a topoisomerase inhibitor which has been shown
to
increase transduction efficiency of rAAV vectors (Proc. Natl. Acad. Sci. USA,
92:5719-
5723, 1995). Forty-eight hours after infection, culture supernatant was
collected and
cells were lysed in 0.5 mL lx lysis buffer (100 mM NaCI, 20 mM Tris pH 7.5, 1
mM
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27
EDTA, O.S% NP40, and O.S% deoxycholate). FGF-2 in the supernatants and lysates
was assayed by ELISA (cat. # DFB00, R & D Systems, Minneapolis, MN) following
manufacturer's instructions. The results are shown below in Table 1.
TABLE 1
S FGF-2 PRODUCTION IN 293 CELLS FOLLOWING INFECTION WITH RAAV-FGF-2
Culture Medium Supernatant (1.5 ml)
sam le infection MOI Eto osideFGF2 rotein
1 0 - <S /ml
2 2X105 - <S /ml
3 2X104 - <S /ml
4 2X103 - <S /ml
1 0 + <S /ml
2 2X105 + 106 /ml ~ 300 /24h/lObcells
3 2X104 + <S /ml
4 2X103 + <S /ml
Cell Lysate (0.5 ml)
sam le infection MOI Eto osideFGF2 rotein
1 0 - 8.95 n /ml __
2 2X105 - 114. n /ml
3 2X104 - 18.8 n /ml
4 2X103 - 11.3 n /ml
1 0 + S.OS n /ml
2 2X105 + 296. n /ml ~ 300n /24h/106
cells
3 2X104 + 48.0 n /ml
4 2X 103 + 13.2 n /ml
EXAMPLE 3
lO CONSTRUCTION OF RAAV VECTORS
A. Construction of pD 10-bFGF-2
The pDlO AAV vector is constructed by replacing the AAV gene
encoding sequences of pD-10 (see Wang, X. et al. J. Virol. 71:3077 (1997),
with the
CMV promoter, multiple cloning site, and BGH polyadenylation sequences from
1 S pKm201 CMV. Briefly, oligonucleotides S'-ggtatttaaa acttgcggcc gcggaatttc
gactctaggc
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28
c-3' (SEQ LD. No. ~ and 5'-gctgcccggg acttgctagc tggatgatcc tccagcgcgg
ggatctcatg
-3' (SEQ LD. No. ~ are used to amplify the CMV expression cassette from
pKm201CMV. The product of this PCR amplification is digested with SmaI and
DraI
and cloned into pD-10 digested with EcoRV. This new vector is named pDlO-CMV.
To construct pD 10-bFGF-2, the synthetic gene for bovine FGF-2 (see
US Patent 5,464,774 for sequence) is digested with EcoRI and SaII, treated
with T4
polymerase to blunt the ends, and then cloned into the StuI site of pDlO-CMV.
The
synthetic gene described above encodes the mature, processed form of bovine
FGF-2.
B. Construction of a rAAV Vector ExpressingyFGF-2-Sc.
pDlO-K-FGF-2-Sc (see Figure 31) was constructed by cloning the FGF-
2 full length humanized bovine cDNA obtained from Scios, into the pDlO vector
backbone containing the kanamycin (Kan) resistance gene. This cDNA contains
the
mutations at amino acid positions 121 and 137 described in example 1. Briefly,
the
FGF-2-Sc cDNA was digested from the parent plasmid with the enzyme Ndel, the
ends
blunted with T4 DNA polymerase, cut with the restriction enzyme HindIII, and
cloned
into the pDlO-CMV vector which had been digested with the enzymes Stul and
HindIII. The nucleotide sequence ofpDlO-K-FGF-2-Sc is illustrated in Figure
32.
C. Infection of Cells with rAAV-FGF-2-Sc Results in the Expression of FGF-2
293 cells were infected as in example 2, with the following
modifications: 4 x 10e5 cells/well were plated in a 12 well dish, and all
wells contained
3 uM etoposide. Three different particle numbers of FGF-2 virus, and a
negative
control CMV-lacZ virus were used to infect the cells. 48 hours after
infection, tissue
culture media was collected and cells lysed in 100 ul lysis buffer containing
Triton-X
100 and CompleteTM protein inhibitor cocktail (Boehringer Mannheim, Germany).
FGF-2 protein in the media and lysates was assayed by ELISA. The results are
shown
below in Table 2 below.
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TABLE 2
FGF-2 PRODUCTION IN 293 CELLS FOLLOWING INFECTION WITH RAAV
D 10-K-FGF-2-S C
Vector Viral Lysates(pg/mL) Culture media
Particles /mL)
D10-K-FGF-2-Sc 1 x 10e10 324492.919 438.621
D10-K-FGF-2-Sc 1 x 10e9 32876.106 46.984
D10-K-FGF-2-Sc 1 x 10e8 6950.039 14.649
CMV -lacz I 1 x 10e10 2327.527 I 17.096
I
EXAMPLE 4
S CONSTRUCTION OF RAAV VECTORS WHICH EXPRESS FGFS AND FGF1 S
A. Cloning FGF-5 into the pDlO-CMV rAAV Vector.
The FGF-S coding region (see U.S. Serial No. 08,602,147) was cloned
into the rAAV pDlO-CMV vector by digestion with the enzymes SacII and XmnI,
resulting in an 814 by fragment. This removed an ORF (ORF-1) upstream of and
overlapping with the FGF-S coding region. The ends of the FGF-5 fragment were
then
blunted with T4 DNA polymerase, and it was cloned into the rAAV pD 10-CMV
vector
linearized with StuI. This vector contains a 1353 by insertion of a
bacteriophage Phi
X174 HaeIII fragment.
The pDlO-CMV-FGF-S vector is illustrated schematically in Figure 3.
1 S In summary, this plasmid contains the CMV immediate/early enhancer +
promoter, the
CMV intron A, an FGF-S coding region, the bovine growth hormone polyA site,
and
AAV ITR sequences. There is a 1353 by insertion of PhiX 174 bacteriophage DNA
cloned into the Notl site between one ITR and the CMV immediate early enhancer
+
promoter region.
B. Packaginu and Functional Analysis of FGF-S rAAV.
rAAV virus was packaged using a triple transfection method as
described in Example 1. However, rather than cesium chloride equilibrium
density
gradient centrifugation, heparin sulfate column chromatography is utilized.
More
specifically, a cell pellet is resuspended in TNM buffer: 20mM Tris pH 8.0,
lSOmM
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NaCI, 2mM MgCl2. Deoxycholic Acid is added to 0.5% to lyse the cells. 50 U/ml
Benzonase is added and the lysed cells are incubated at 37 degrees to digest
any nucleic
acids. The cell debris is pelleted and the supernatant is filtered through a
0.45um filter
and then a 0.22um filter. The virus is then loaded onto a 1.5m1 Heparin
sulfate column
5 using the Biocad HPLC. The column is then washed with 20mM Tris pH 8.0,
100mM
NaCI. The rAAV particles are eluted with a gradient formed with increasing
concentrations of NaCI. The fractions under the peak are pooled and filtered
through a
0.22um filter before overnight precipitation with 8% PEG 8000. CaClz is added
to
25mM and the purified particles are pelleted and then resuspended in HBS#2:
150mM
10 NaCI, SOmM Hepes pH7.4.
Briefly, 8 x 108 or 8 x 109 particles of the resulting FGF-5 virus were
used to infect 293 cells, which were simultaneously treated with 3 uM
etoposide to
enhance viral expression levels. At 24 hours post-infection, tissue culture
media and
cell lysates were harvested and analysed by Western blotting. Briefly, protein
samples
15 were run on a 4-20% tris-glycine gradient gel, and transferred to
nitrocellulose by
standard procedures. After blocking with 5% milk in PBS, the membrane was
incubated with an anti-human FGF-5 antibody (R and D systems, made in goat) at
a
dilution of 1:1,000 for one hour at room temperature. After the membrane was
washed
3 times in PBS + 0.05% Tween-20, it was incubated with an anti-goat secondary
20 antibody conjugated to peroxidase (1:5,000 dilution). The membrane was then
washed
and the FGF-S protein detected by chemiluminescence.
Results of the Western blot are shown in Figure 4. Briefly, lane 1
represents 50 ng of the 29.5 Kd recombinant FGF-5 protein (R and D systems).
Lane 2,
media from cells infected with 8 x 109 viral particles and treated with
etoposide, shows
25 no FGF-S expression. Lane 3 is an uninfected cell lysate control. Lane 4
and 5 are
lysates from cells infected with 8 x 108 or 8 x 109 viral particles,
respectively, and Lanes
6 and 7 are lysates from cells infected with 8 x 108 or 8 x 109 viral
particles and treated
with 3 uM etoposide. Lanes 4-7 all show positive FGF-S expression. Lane 8 is a
negative control of lysate from uninfected cells.
30 In summary, although the FGF-5 signal sequence was intact, FGF-5
protein was detected in the cell lysate only.
C. Cloning FGF-5 Lacking a Si nag 1 Sequence, into rAAV pDIO-CMV.
Oncogenic activity is associated with the wild-type FGF-5 molecule
(Zhan et al., 1988; Bates et al., 1991). To improve its safety, the codons for
the first 21
amino acids of FGF-5's signal sequence were removed by PCR amplification of
the
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31
above pD 10-CMV-FGF-5 plasmid with the following primers:
AGA/TAT/AAG/CTT/AC C/ATG/GGT/GAA/AAG/CG T/CTC/GCC/CCC/AAA (5',
SFGFMUTB; SEQ LD. No.~ and CGC/GCG/CTC/GAG/AC
C/ATG/AGG/AAT/ATT/AT C/CAA/AGC/GAA/ACT (3', 3FGFSWT; SEQ LD. No.
~. The 5' primer contains point mutations which destabilize G/C rich hairpin
structures of the FGF-5 mRNA, and should increase levels of gene expression.
The
PCR product was digested with HindIII and XhoI (restriction sites introduced
by the
primers), and cloned by standard methods, into the pDlO vector digested with
the same
enzymes. The pD 10-CMV-FGF-5 (sig-) vector is illustrated schematically in
Figure 5.
In summary, the pDlO-CMV-FGF-5 (sig-) plasmid contains the CMV
immediate/early enhancer + promoter, the CMV intron A, the FGF-5 coding region
with the modifications described in Example C above, the bovine growth hormone
polyA site, and the AAV ITR sequences. There is a 1353 by insertion of PhiX
174
bacteriophage DNA clones into the Notl site between one ITR and the CMV
immediate
early enhancer + promoter region.
D. Western Analysis of 293 Cells Transfected with pDlO-CMV-FGF-5 (sig=).
Expression of FGF-5 protein was demonstrated by transient transfection
of 293 cells with the plasmid pDlO-CMV-FGF-S (sig-), by standard methods.
After 48
hours, tissue culture media and cell lysates were harvested. Western analysis
was
performed with an anti-human FGF-5 antibody (R and D systems) as described
above.
Results of the Western analysis are provided below in Figure 6. Briefly,
lane 1 represents 50 ng of the 29.5 Kd recombinant FGF-5 prot~~°rr. (R
and D systems).
Lanes 2 and 3, showing FGF-5 expression, are cell lysates from 293 cells
transfected
with two different clones of the pDlO-CMV-FGF-5 sig- plasmid. Lane 4 is lysate
from
cells transfected with a negative control plasmid CMV-Epo. Lanes 5, 6 and 7
represent
media from cells transfected with different clones of the pDlO-CMV-FGF-5 sig-
plasmid, respectively, and the CMV-Epo plasmid. As is evident from this
figure, FGF-
5 protein was detected in the cell lysate only.
E. Generation of FGF-5 (signal -) rAAV.
FGF-5 (sig-) rAAV virus is packaged using the triple transfection
method described in more detail above.
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F. Clonin FGF-18 into the pDlO-CMV rAAV Vector.
The FGF-18 coding region (see U.S. Provisional Application Serial No.
60/083,553) was cloned into the pDlO-CMV vector as a PstI to EcoRV fragment,
using
restriction sites found in both the FGF-18 and the multiple cloning site of
the pDlO-
CMV vector. The vector contains a 1353 by insertion of PhiXl74 bacteriophage
DNA
(see Example A).
A schematic illustration of pDlO-CMV-FGF-18 is provided in Figure 7.
Briefly, this plasmid contains the CMV immediate/early enhancer + promoter,
the CMV
intron A, the FGF-18 coding region, the bovine growth hormone polyA site, and
the
AAV ITR sequences. There is a 1353 by insertion of PhiX 174 bacteriophage DNA
cloned into the Notl site between one ITR and the CMV immediate early enhancer
+
promoter region.
G. Analysis of 293 Cells Transfected with pDlO-CMV-FGF-18 Plasmid.
Expression of FGF-18 protein was assessed by transient transfection of
293 cells followed by Western analysis, using standard methods. Cell lysates
and tissue
culture media were harvested at 48 hours post transfection. An anti-peptide
FGF-18
rabbit polyclonal antibody, generated against a selected polypeptide from
recombinant
FGF-18, was used at a dilution of 1:2,500 for one hour at room temperature.
The
secondary antibody, an anti-rabbit IgG conjugated to peroxidase, was used at a
dilution
of 1:25,000.
Results of the Western analysis are provided in Figure 8. Briefly, lanes
1-3 represent 1, 2 and 10 ul of tissue culture media from cells transfected
with the
pDlO-CMV-FGF-18 plasmid. Lane 4 is blank. Lanes 5, 6 and 7 contain 2, 10 and
20
ul of lysate from the transfected cells. Lanes 8 and 9 are negative controls;
20 ul of
tissue culture media and cell lysate, respectively, from uninfected cells.
Lane 10
contains a positive control; an FGF-18-maltose binding protein fusion ((MBP);
predicted size = 80 Kd, larger than the FGF-18 protein).
H. Packaging of the pDIOCMV-FGF-18 plasrnid into rAAV particles
FGF-18 rAAV virus was generated by the triple transfection method.
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EXAMPLE 5
AAV - LACZ INJECTED RETINA
A. Subretinal imiection of rAAV
Albino Sprague-Dawley rats were injected at 14 -15 days postnatal
(P 14- P 15). Animals were anesthetized by ketamine/xylazine inj ection, and a
local
anesthetic (proparacain HCl) was applied topically to the cornea. An aperture
was
made through the inferior cornea of the eye with a 28 gauge needle. Subretinal
injections of 2-3~1 of AAV-CMV-Lac-Z were then made by inserting a blunt 32
gauge
needle through the opening and delivering the rAAV suspension into the
subretinal
space in the posterior retina. The contralateral eye was either uninfected,
injected
subretinally with PBS, or with a control rAAV containing a reporter gene.
B. Staining Protocol
Cryosections of the retina were stained with Bluo-gal for b-galactosidase
reaction product of lacZ. In all wild type rats tested (3), positive staining
was visible in
the interior of the whole eyecup upon gross examination (see Figure 9). 100pm
thick
agarose or 20~.m thick cryosections of retinas indicated that most of the b-
gal positive
staining localized to the photoreceptors. There were a small number of LacZ
positive
retinal ganglion cells observed.
C. Anti-b-galactosidase immunocytochemistry
Sections from 3 wildtype and 2 transgenic rats were stained with a
polyclonal antibody against b-galactosidase. These results were comparable to
the
bluo-gal results, primarily demonstrating photoreceptor-specific staining. Two
out of
five rats showed no positive staining.
D. Results
Subretinal injection of 2 ul of AAV-CMV-lacZ effectively transduced a
large number of photoreceptor and retinal pigment epithelial (rpe) cells
following a
single intraocular inoculation of AAV-CMV-lacZ into the subretinal space (SRS)
of the
rat eye. The lateral extent of lacZ reporter gene expression was typically 1/3
to 1/2 of
the retinal expanse following a single AAV-CMV-LacZ injection. This finding
was
confirmed by bluo-gal staining of the b-galactosidase reaction product of the
lacZ gene
as well as by immunocytochemistry using an antibody specific for b-
galactosidase. The
AAV-CMV-lacZ vector was effective at transducing photoreceptor and RPE cells
in
both the normal (wildtype) and affected, degenerating (transgenic) rat retina.
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EXAMPLE 6
RETINAL TISSUE ANALYSIS OF RAAV-FGF-2 INFECTED CELLS, VS. CONTROLS
A. Subretinal ini ection of rAAV
Line 3 albino transgenic rats (P23H-3) on an albino Sprague-Dawley
background (produced by Chrysalis DNX Transgenic Sciences, Princeton, NJ) were
injected at the ages of P14 or P15. Animals were anesthetized by
ketamine/xylazine
injection, and a local anesthetic (proparacain HCl) was applied topically to
the cornea.
An aperture was made through the inferior cornea of the eye with a 28 gauge
needle.
The subretinal injections of 2 ~l were then made by inserting a blunt 32 gauge
needle
through the opening and delivering the rAAV suspension into the subretinal
space in the
posterior retina. The intent was to inject into the subretinal space of the
posterior
superior hemisphere, but we sometimes found histologically that the injection
site was
located just inferior to the optic nerve head. The opposite eye was either
uninfected,
injected subretinally with PBS, with control rAAV containing no neurotrophin
or
1 S containing proteins not known to possess neurotrophic properties.
B. Histopathology Protocol / Retinal tissue anal
The rats were euthanized by overdose of carbon dioxide inhalation and
immediately perfused intracardially with a mixture of mixed aldehydes (2%
formaldehyde and 2.5 % glutaraldehyde). Eyes were removed and embedded in
epoxy
resin, and 1 ~m thick histological sections were made along the vertical
meridian.
Tissue sections were aligned so that the ROS and Miiller cell processes
crossing the
inner plexiform layer were continuous throughout the plane of section to
assure that the
sections were not oblique, and the thickness of the ONL and lengths of RIS and
ROS
were measured as described (LaVail, et al) . Briefly, 54 measurements of each
layer or
structure were made at set points around the entire retinal section. These
data were
either averaged to provide a single value for the retina, or plotted as a
distribution of
thickness or length across the retina. We also compared the greatest 3
contiguous
values for ONL thickness in each retina, to determine if any region of retina
(e.g.,
nearest the injection site) showed proportionally greater rescue; although
most of these
values were slightly greater than the overall mean of all 54 values, they were
no
different from control values than the overall mean. Thus, the overall mean
was used in
the data cited, since it was based on a much larger number of measurements.
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C. Results
Two surgical methods of delivery of rAAV-CMV-FGF2 were
completed; intravitreal and subretinal injection.
1. Intravitreal inj ection
5 RAAV-CMV-FGF-2 was injected into the right eye of nine transgenic
S334ter rats after day P15 (the left eye was not injected). 5334(4) transgenic
animals
were used to assess the rescue effect of rAAV-CMV- FGF-2 on degenerating
photoreceptor cells when delivered by intravitreal injection. The rats were
all sacrificed
at age p60 and the embedded in plastic and sectioned to assess morphology and
10 therapeutic effect as assayed by the preservation of thickness of outer
nuclear layer.
Superior and inferior regions of eyecup are quantitated by measuring the ONL
thickness
using a BioQuant morphometric measuring system (BioQuant). Injected eyes were
evaluated along with uninfected control eyes.
15 Control Left superior - 16.52 +/- 2.77 um
Inj ected Right superior - 19.71 +/- 5.27 um
Control Left inferior - 22.64 +/- 2.11 um
Injected Right inferior - 26.47 +/- 3.55 um
Based upon these results it was evident that there is a rescue effect of
AAV-CMV-FGF-2 when delivered intraocularly into the vitreous cavity.
2. Subretinal injection of rAAV-CMV-FGF-2
Experiment A. - Location of injection - subretinal , 7 rats-both right and
left eyes injected, 3 rats-(left eye = uninfected). Number of rats injected -
10 rats all
wild type p15 on day of injection. One rat was sacrificed every week starting
at week 2.
Expression of FGF-2 was assessed, as well as any signs of inflammation or
neovascularization.
Experiment B. - Location of injection - subretinal, 5 rats- right eyes
injected w/vector left eyes injected with PBS, 4 rats- right eyes injected
w/vector (left
eye = uninfected). Number of rats injected - 11 transgenic 5334(4) rats - all
were p15
on day of injection. The rats were sacrificed at age p60 and the embedded in
plastic and
sectioned to assess histopathology and number of surviving photoreceptor
cells. .
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Anatomic indication of therapeutic effect (photoreceptor rescue) was
assessed histologically. Briefly, eyes injected with rAAV-CMV-FGF2 retained
significantly more photoreceptors at P60, P75 and P90 than uninfected
contralateral
control eyes of the same animal. Retinas receiving a subretinal injection of
AAV-
CMV-FGF2 at P14-15 retained 71% of the normal ONL thickness, compared to about
47% in the uninfected controls (see Figures 11, 12, 13 and 14).
There was little or no rescue in PBS-injected control eyes (p>0.169 in all
cases). This is consistent with previous reports that needle injury to the
retina in young
rats (P 14-P 15) does not rescue photoreceptors or up-regulate bFGF mRNA
expression.
3. Subretinal infection of rAAV-CMV-FGF-2
Two to three microliters of rAAV-CMV-FGF-2 vector was injected into
the subretinal space between the photoreceptors and the adjacent retinal
pigment
epithelium at P14 or P15. Rats were sacrificed and eyes examined at time
points
between P60-P90. At these ages in uninfected control eyes of S334ter rats, the
ONL
thickness, which is an index of photoreceptor cell number, was reduced to
about 60% of
normal.
Evidence of anatomic rescue was found to be significant to the p=.005
confidence level in retinas transfected by rAAV-CMV-FGF-2 when compared to the
control AAV vectors or sham injection of PBS by ANOVA (analysis of Variance
statistical measures). JMP statistical analysis software (Copyright (c) 1999
SAS
Institute Inc. Cart', North Carolina, USA).
EXAMPLE 7
ANTIBODY STAINING OF RAAV-FGF-2 INFECTED CELLS
A. Injection Protocol
Albino Sprague-Dawley rats were injected with rAAV-CMV-FGF-2 at
the ages of P14 or P15 essentially as follows. Briefly, wild-type animals were
anesthetized by ketamine/xylazine injection, and a local anesthetic
(proparacain HCl)
was applied topically to the cornea. An aperture was made through the inferior
cornea
of the eye with a 28 gauge needle. The subretinal injections of 2-3 ul of rAAV-
CMV-
FGF-2 were then made by inserting a blunt 32 gauge needle through the opening
and
delivering the rAAV suspension into the subretinal space in the posterior
retina. The
contralateral eye was either uninfected, injected subretinally with PBS, wild-
type
rAAV, or with rAAV-CMV-lacZ.
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B. Staining Protocol
Fixed eyecups were embedded in OCT and cryosectioned in 20um thick
sections. Sections from 10 wt rats were stained with antibody to FGF-2.
(primary- anti
FGF-2 1:500 (commercial antibody purchased from R&D systems) (secondary-anti
S goat Cy3 conjugate (Sigma, St. Louis. MO)
C. Results
Immunohistochemistry was used to look for expression of FGF-2 in the
eye. Two rats were examined every week starting at 3 weeks post-injection.
Retinas
were examined for expression of FGF-2 and also examined histopathologically
for
signs of inflammation or neovascularization.
Results are shown in Figure 15. Briefly, expression of FGF-2 was found
in retinal photoreceptor cells as well as RPE cells at 3S days following
inoculation with
2-3 ul of rAAV-CMV-FGF-2. Less significant expression was noted in retinal
bipolar
interneurons and retinal ganglion cells (RGCs) following injection into the
subretinal
1 S space (SRS). No significant staining above background was observed in
sections
injected with PBS or rAAV-CMV-lacZ vectors.
EXAMPLE 8
RETINAL TISSUE ANALYSIS OF RAAV-FGF-S AND -18 INFECTED CELLS, VS. CONTROLS
A. Subretinal injection of rAAV
Line 4 albino transgenic rats (S334ter-4) on an albino Sprague-Dawley
background (produced by Chrysalis DNX Transgenic Sciences, Princeton, NJ) were
injected at age P15. Animals were anesthetized by ketamine/xylazine injection,
and a
local anesthetic (proparacain HCl) was applied topically to the cornea. An
aperture was
made through the inferior cornea of the eye with a 28 gauge needle. The
subretinal
injections of 2.S ~l were then made by inserting a blunt 32 gauge needle
through the
opening and delivering the rAAV suspension into the subretinal space in the
posterior
retina. The opposite eye was either uninfected, injected subretinally with
PBS, with
control rAAV containing no neurotrophin or containing proteins not known to
possess
neurotrophic properties.
B. Histopathology Protocol / Retinal tissue analysis
The rats were euthanized by overdose of carbon dioxide inhalation and
immediately perfused intracardially with a mixture of mixed aldehydes (2%
formaldehyde and 2.5 % glutaraldehyde). Eyes were removed and embedded in
epoxy
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resin, and 1 ~m thick histological sections were made along the vertical
meridian.
Tissue sections were aligned so that the ROS and Muller cell processes
crossing the
inner plexiform layer were continuous throughout the plane of section to
assure that the
sections were not oblique, and the thickness of the ONL was measured as
described
(LaVail, et al 19XX). Briefly, 54 measurements of each layer or structure were
made at
set points around the entire retinal section. The 27 measurements from the
inferior
region and the superior region of the retina were averaged separately to give
two values
for each eye. This separation was made because the retina degenerates at
different rates
in these two regions of the S334ter-4 animal model.
C. Results
Sub-retinal injections of both rAAV-CMV-FGF-5 and rAAV-CMV-
FGF-18 were performed.
1. Sub-retinal injection of rAAV-CMV-FGF-5
Experiment. - Location of injection - subretinal, 3 rats- right eyes
injected w/vector left eyes injected with PBS, 8 rats- right eyes injected
w/vector left
eyes injected with rAAV-CMV-LacZ, 4 rats- right eyes injected w/vector (left
eye =
uninfected). Number of rats injected - 15 transgenic S334ter-4 rats - all were
p15 on
day of injection. The rats were sacrificed at age p60. The retinas were
embedded in
plastic and sectioned to assess histopathology and number of surviving
photoreceptor
cells.
Anatomic indication of therapeutic effect (photoreceptor rescue) was
assessed histologically. The injection of rAAV-CMV-FGF-5 resulted in
significant
rescue of photoreceptors, compared to PBS injected, rAAV-CMV-LacZ injected,
and
uninfected eyes (see Figure 33). The rescue was significant to the p=.OS
confidence
level for all three comparisons, by ANOVA (analysis of Variance statistical
measures).
JMP statistical analysis software (Copyright (c) 1999 SAS Institute Inc.
Cart', North
Carolina, USA).
2. Sub-retinal injection of rAAV-CMV-FGF-18
Experiment. - Location of injection - subretinal, 3 rats- right eyes
injected w/vector left eyes injected with PBS, 3 rats- right eyes injected
w/vector left
eyes injected with rAAV-CMV-LacZ, 4 rats- right eyes injected w/vector (left
eye =
uninfected). Number of rats injected - 10 transgenic S334ter-4 rats - all were
p15 on
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day of injection. The rats were sacrificed at p60, and the retinas embedded in
plastic
and sectioned.
Eyes injected with rAAV-CMV-FGF-18 retained significantly more
photoreceptors at P60 than PBS injected, rAAV-CMV-LacZ injected, or uninfected
control eyes. Each comparison, by ANOVA, was statistically significant to the
p=.OS
confidence level.
EXAMPLE 9
IN VIVO DELIVERY TO RETINAL GANGLION CELLS (RGCS)
A. Intra-vitreal injection of AAV vectors
All surgical procedures were performed in female adult Sprague-Dawley
rats (180-200 g; Charles River Breeders) under general anesthesia (7% chloral
hydrate;
0.42 mg per g of body weight, i.p.) in accordance with the Use of Animals in
Neuroscience Research and McGill University Animal Care Committee guidelines
for
the use of experimental animals.
Briefly, rAAV-CMV-lacZ (5 ~tl; see above) was injected into the
vitreous chamber in the superior (dorsal) hemisphere of the retina using a
posterior
approach as described (Di Polo, PNAS, 1998). Control eyes were injected with
equal
volumes of Hepes-buffered saline (HBS, virus vector).
B. Identification of RPCs
For visualization of RGCs, neurons were retrogradely labeled with the
fluorescent tracer Fluorogold (Fluorochrome, Englewood, CO) at 2% in 0.9% NaCI
containing 10% dimethyl sulfoxide by application of the tracer to both
superior colliculi
7 days prior to analyses as described (Vidal-Sanz et al., 1988). Anesthetized
rats were
then perfused intracardially with 4% paraformaldehyde in 0.1 M phosphate
buffer (PB,
pH 7.4) and the eyes were immediately enucleated. The anterior part of the eye
and the
lens were removed and the remaining eye cup was immersed in the same fixative
for 2
hr at 4°C. Eye cups were cryoprotected in graded sucrose solutions (10-
30% in PB) for
several hours at 4°C, embedded in O.C.T. compound (Tissue-Tek, Miles
Laboratories,
Elkhart, IN) and frozen in a 2-methylbutane/liquid nitrogen bath. Retinal
radial
cryosections (12-15 Vim), obtained along the vertical meridian of the eye,
were collected
onto gelatin-coated slides and processed for immunocytochemistry.
Alternatively, entire
eyes were rinsed three times (15 min each) in PBS at room temperature with
gentle
shaking, to whole-mount histochemical staining as described below.
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C. Histochemical Analysis
Expression of the bacterial LacZ gene in whole retinas was detected by
standard histochemical staining reactions using halogenated indoyl-13-D-
galactoside
(Bluogal; GIBCO BRL). Following removal of the anterior eye structures and
lens, eye
5 cups were incubated overnight in a staining solution containing SmM K-
ferricyanide, 5
mM K-ferrocyanide, 2 mM MgClz, and 0.5 mg/ml Bluo-gal at 37°C. Retinas
were then
dissected, fixed for an additional 30 min and flat-mounted vitreal side up on
glass
slides.
For visualization of the AAV-mediated lacZ gene product in retinal
10 radial sections, cryosections were incubated in 10% normal goat serum (NGS)
in 0.2%
Triton X-100 (Sigma, St. Louis, MO) in phosphate buffer saline (PBS) for 30
min at
room temperature to block non-specific binding. Two primary antibodies raised
against
the lacZ gene product were used with similar results. A polyclonal anti-
betagal
antibody (diluted 1:1000; 5 prime-~3 prime, Inc., Boulder, CO) and a
monoclonal anti-
15 LacZ antibody (diluted 1:500; Promega, Madison, WI). Primary antibodies
were added
in 2% NGS in 0.2% Triton X-100 and incubated overnight at 4°C. Sections
were
subsequently processed with anti-rabbit Cy3-conjugated IgG (diluted 1:500,
Jackson
Immunoresearch, West Grove, PA) or anti-mouse Cy3-conjugated IgG (diluted
1:500,
Jackson Immunoresearch) and mounted. Control sections were treated in the same
way
20 but with omission of the primary antibody. Sections were visualized by
fluorescent
microscopy (Polyvar, Reichert-Jung).
Expression of the heparan sulfate proteoglycan receptor in the retina was
examined using a monoclonal anti-heparan sulfate antibody (He:~ ;'S-1, diluted
1:1,000,
Seikagaku Corporation, Tokyo, Japan). Following overnight incubation at
4°C, sections
25 were processed with biotinylated anti-mouse Fab fragment (Jackson
Immunoresearch),
avidin-biotin-peroxidase reagent (ABC Elite Vector Labs, Burlingame, CA),
followed
by reaction in a solution containing 0.05% diaminobenzidine tetrahydrochloride
(DAB)
and 0.06% hydrogen peroxide in PB (pH 7.4) for 5 min. For analysis of co-
localization
of heparan sulfate in Fluorogold-labeled neurons, sections were processed with
Cy3-
30 coupled anti-mouse IgG (Jackson Immunoresearch) after incubation in primary
antibody. In all cases, the primary antibody was omitted in control sections.
Sections
were mounted and visualized by light or fluorescent microscopy.
Quantification of AAV-transduced cells in the ganglion cell layer of
retinal flat-mounts was performed in two ways: i) by counting the entire
number of
35 Bluo-gal positive cells in each of the retinal quadrants (superior,
inferior, temporal and
nasal); and ii) by counting the number of cells in three standard areas (at l,
2 and 3 mm
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from the optic disc) of each quadrant as previously described (Villegas-Perez
et al.,
1993).
For quantification of Fluorogold, Bluo-gal or HepSS-1 positive cells in
retinal radial sections, the entire number of labeled cells per section was
counted under
fluorescent microscopy. Four to five serial sections per eye were routinely
counted and
a mean value per animal was obtained, followed by the calculation of a mean
value for
the entire experimental group which consisted of 4-5 rats. Results were
analyzed using
the Sigmastat program (Jandel, San Rafael Madera, CA) by a student's t test
(paired
groups).
D. Results
Analysis of retinas from eyes that received a single intravitreal injection
of rAAV-CMV-lacZ demonstrated a large number of LacZ-positive cells throughout
the
entire GCL as assessed by histochemical LacZ staining of both flat-mounts
(Figure
16A) and radial sections (Figure 16B). In many cases, RGCs transduced by AAV
could
be unequivocally identified because the LacZ reaction product filled their
axons that
converged at the level of the optic nerve head. In addition, LacZ-positive
photoreceptor
nuclei were observed but were always restricted to the vicinity of the
injection site (not
shown). No staining was observed in control eyes injected with virus vector.
No signs
of cytotoxic damage or cellular immune reaction to the viral vector were
observed in
any of the retinas examined.
Quantification of LacZ-positive cells in the GCL of retinal flat-mounts
demonstrated a 2.8-fold increase between 2 and 4 weeks after intravitreal
injection of
the rAAV-CMV-lacZ vector (Figure 17). For example, we found 27,5697,646
cells/retina (mean~S.D.;n=3) and 79,04310,321 cells/retina (n=4) expressing
the LacZ
gene product at 2 and 4 weeks after intraocular administration of the vector,
respectively. A comparable number of cells expressing the AAV-mediated
transgene at
4 weeks was observed at 8 weeks (70,22112,500; n=3) following rAAV-CMV-lacZ
injection. Although the majority of LacZ-positive cells were observed in the
superior
hemisphere at 2 weeks after virus administration, there was robust transgene
expression
throughout the entire retina by 4 and 8 weeks as assessed by quantification of
Lac-Z
positive cell densities in all retinal quadrants at these time points (Figure
17).
To identify the cellular localization of the AAV-mediated LacZ gene
product, we combined immunocytochemical staining of LacZ with retrograde
tracing of
RGC bodies using Fluorogold backlabeling from the superior colliculi. Double-
labeling
experiments indicated that the majority of cells in the GCL expressing the
LacZ gene
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product were also Fluorogold-positive (Figure 18). Analysis of the number of
Fluorogold-labeled cells in the GCL expressing the LacZ gene product indicated
that
30029 (mean~S.D.; n=4) expressed both markers out of 33032 Fluorogold-labeled
cells (the average RGC population per retinal radial section). This indicates
that ~92%
of RGCs, identified by the Fluorogold label, also expressed the AAV-mediated
LacZ
gene product (Figure 19). In all retinas examined, we routinely observed a
number of
Lac-Z positive cells that were not labeled with Fluorogold (Figures 18 and
19). Thus, it
is possible that these cells are displaced amacrine cells or RGCs that failed
to
incorporate the retrograde tracer. Together, these results indicate that RGCs
are
preferentially transduced by recombinant AAV following intravitreal injection
of this
viral vector.
To investigate the molecular mechanisms underlying preferential
transduction of RGCs by AAV, we first examined the expression of the heparan
sulfate
proteoglycan, which mediates both AAV attachment and infection of target cells
(Summerford et al., 1998), in the adult retina. Immunostaining of retinal
radial sections
with a specific antibody against heparan sulfate (HepSS-1) demonstrated robust
staining
in the GCL (Figure 20). Positive immunolabeling was clearly visualized in both
neuronal somata and axonal bundles in the fiber layer. More diffuse and sparse
staining
was observed in some photoreceptor nuclei and cells in the inner nuclear
layer. No
staining was observed in control retinal sections in which the primary
antibody was
omitted (not shown).
To determine the cell type within the GCL that express the heparan
sulfate proteoglycan receptor, we performed a double-labeling study in which
RGCs
were first retrogradely labeled from the superior colliculi followed by
immunostaining
of retinal sections with HepSS-1. Our analysis showed that 29923
(mean~S.D.;n=4)
cells in the GCL expressed both Fluorogold and HepSS-1 markers out of 31534
Fluorogold-labeled cells which represent the average RGC population visualized
per
retinal radial section. The large population of RGCs (~95%) expressing heparan
sulfate
proteoglycan receptor correlated well with the number of RGCs expressing the
AAV-
mediated transgene product (~92%). Together, these data suggest that
preferential
transduction of adult RGCs by recombinant AAV is mediated by the heparan
sulfate
proteoglycan receptor expressed by these neurons.
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EXAMPLE 10
CONSTRUCTION OF A RAAV VECTOR EXPRESSING
VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) 16S
S The human VEGF-16S cDNA was cloned from the PCR-Blunt II Topo
Vector (Invitrogen) into the pDlO-CMV rAAV vector as an EcoRl fragment (pDlO-
VEGFuC). The pDlO-VEGFUC vector is illustrated schematically in Figure 21, and
its
nucleotide sequence is shown in Figure 22. The VEGFUC rAAV virus was packaged
using the triple transfection method and purified by column chromatography.
Briefly, a cell pellet is resuspended in TNM buffer: 20mM Tris pH 8.0,
lSOmM NaCI, 2mM MgCl2. Deoxycholic Acid is added to O.S% to lyse the cells.
SOu/ml Benzonase is added and the lysed cells are incubated at 37 degrees to
digest any
nucleic acids. The cell debris is pelleted and the supernatant is filtered
through a
0.4Sum filter and then a 0.22um filter. The virus is then loaded onto a l.Sml
Heparin
1 S sulfate column using the Biocad. The column is then washed with 20mM Tris
pH 8.0,
100mM NaCI. The rAAV particles are eluted with a gradient formed with
increasing
concentrations of NaCI. The fractions under the peak are pooled and filtered
through a
0.22um filter before overnight precipitation with 8% PEG 8000. CaCl2 is added
to
2SmM and the purified particles are pelleted and then resuspended in HBS#2:
lSOmM
NaCI, SOmM Hepes pH7.4.
EXAMPLE 11
INFECTION OF 293 CELLS WITH D1O-VEGF RAAV RESULTS IN
VEGF PROTEIN EXPRESSION
2S The functionality of the viral particles was assessed by infection of 293
cells with 3 different viral multiplicities of infection (MOIs); 1 x 10e7, 1 x
10e8 and 1 x
10e9 viral particles per 4 x lOeS 293 cells, in the presence of l.S uM
etoposide. At 48
hours post infection, tissue culture media (sups) and cell lysates were
harvested. VEGF
protein levels were determined using a Quantikine human VEGF sandwich ELISA
kit
(R and D Systems, see Figure 23). VEGF protein concentrations are given in
pg/ml.
The two highest MOIs gave values significantly above that of the cells
infected with a
negative control virus. The levels of secreted VEGF were approximately 4-7
fold
higher than those of the lysates.
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EXAMPLE 12
INFECTION OF RETINAL PIGMENT EPITHELIAL (RPE) CELLS WITH D1 O-VEGF RAAV
RESULTS IN VEGF PROTEIN SECRETION
S VEGF expression levels in a monolayer of cultured primary (or very
early passage) human fetal RPE cells infected with D10-VEGF 165 rAAV were
clearly
elevated relative to endogenous levels. Cells were infected with a range of
rAAV
particles from 0 to 1 x lOeS per cell. VEGF expression was dose dependent,
increased
over time, and secretion appeared to be somewhat higher from the apical
surface. In a
representative experiment, RPE cells infected with 1 x lOeS viral particles
secreted >
100 ng/1 x 10e6 cells from the apical surface and SO ng/1 x 10e6 cells from
the basal
surface at 8 days post infection. The polarity of VEGF secretion from human
fetal RPE
cells infected with 3 different MOIs is shown in Figure 24.
EXAMPLE 13
1 S INFECTION OF RETINAL PIGMENT EPITHELIAL (RPE) CELLS WITH VEGF RAV RESULTS
IN VEGF PROTEIN SECRET10N AND DECREASED MEMBRANE CONDUCTANCE
Infection of cultured human fetal RPE cells with recombinant VEGF
adenovirus (AV) results in secretion of very high levels of VEGF from both the
apical
and basal surfaces of the RPE. MOTs of 0 to 1,000 or 0 to 10,000 particles per
cell
were used in two separate experiments. In both cases, expression levels
increased over
time, peaking at approximately 100-200 ug/1 x 10e6 cells at wee highest MOTs
(see
Figure 2S). In addition, the total transepithelial membrane resistance of the
RPE
monolayer decreased significantly at all MOIs, and by approximately 4-S fold
at the
2S highest MOIs (see Figure 26).
EXAMPLE 14
CONSTRUCTION OF A RAAV VECTOR EXPRESSING SOLUBLE FLT-1 (SFLT-1) RECEPTOR
The sFlt-1 cDNA was cloned from the Blunt II Topo Vector (Invitrogen)
into the pDlO-CMV rAAV vector as an EcoRl fragment (pDlO-sFlt-1). The pDlO-
sFlt-1 vector is illustrated schematically in Figure 27, and its nucleotide
sequence is
shown in Figure 28. The human sFlt-1 rAAV virus was packaged using the triple
transfection technique and purified by column chromatography.
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EXAMPLE 15
IN VITRO ASSAY FOR ANTI-ANGIOGENIC ACTIVITY
This example describes the HUVEC (human umbilical vein endothelial
5 cell) proliferation assay, which can be utilized to determine the anti-
angiogenic activity
of a molecule (see generally, Gerritsen et al, 1999. JBC 274:9116-9121).
Briefly,
HUVEC cells are seeded on collagen-coated 96-well plates at 6,000 cells/cmZ in
Clonetics EGM media. EGM media contains endothelial cell growth supplements,
10%
fetal bovine serum, 2mM L-glutamine, and antibiotics. Cells are allowed to
attach for 4
10 hours. Medium is then replaced with 40 ng/ml bFGF, 40 ng/ml VEGF, and 80 nM
PMA in 1X basal medium consisting of M199 supplemented with 1% fetal bovine
serum, 1X ITS, 2 mM L-glutamine, 50 ug/ml of ascorbic acid, 26.5 mM NaHC03,
and
appropriate concentration of antibiotics. Cells are cultured in the above
medium in the
presence of the sample supernatant or vector for 4 hours. The sample
supernatant
15 would come from 293 cells transfected with the appropriate plasmid
construct
expressing the molecule we are testing for anti-angiogenic activity. Finally,
5 uL
(100uM) of 5-bromo-2'-deoxyuridine (BrdU) is added in a final volume of 100
uL/well,
and cells were incubated for another 20 hours. BrdU incorporation was
evaluated by an
enzyme-linked immunosorbent assay kit from Boehringer Mannheim.
From the foregoing, it will be appreciated that, although specific
embodiments of the invention have been described herein for purposes of
illustration,
various modifications may be made without deviating from the spirit and scope
of the
invention. Accordingly, the invention is not limited except as by the appended
claims.
