Post on 03-Apr-2023
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A HYPOXIA RESPONSIVE GLIAL CELL-SPECIFIC GENE THERAPY VECTOR FOR 1
TARGETING RETINAL NEOVASCULARIZATION 2
Manas R Biswal,1 Howard M Prentice,2,3 C Kathleen Dorey,4 and Janet C Blanks2 3
1Integrative Biology Ph.D. Program, 2Center for Complex Systems and Brain Sciences, 4
Charles E. Schmidt College of Science, 3Charles E. Schmidt College of Medicine, 5
Florida Atlantic University, 4 Virginia Tech Carilion School of Medicine. 6
Supported by NIH Grant EYO16119 (JCB), American Heart Association grant 7
0815022E (MRB), Seed grant from Neuroscience Research Priority Grant funded by 8
Florida Atlantic University (JCB and HMP) 9
Word Count (Abstract: 248 words, Manuscript: words 3267 without figure legends) 10
Corresponding author: Janet C. Blanks, Center for Complex Systems and Brain 11
Sciences, Florida Atlantic University, 777 Glades Road, Boca Raton, FL 33431. Email: 12
blanks@fau.edu 13
Preliminary data was presented in abstract form at Society for Neuroscience, San 14
Diego, CA 2011. 15
Short title: Hypoxia-regulated gene therapy in retina 16
Key Words: Hypoxia, GFAP, Müller cell, HIF-1 responsive element, OIR model, gene 17
therapy 18
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IOVS Papers in Press. Published on November 6, 2014 as Manuscript iovs.14-13932
Copyright 2014 by The Association for Research in Vision and Ophthalmology, Inc.
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ABSTRACT: 20
Purpose: Müller cells, the major glial cell in the retina, play a significant role in retinal 21
neovascularization in response to tissue hypoxia. We previously designed and tested a 22
vector using a hypoxia-responsive domain and a glial fibrillary acidic protein (GFAP) 23
promoter to drive green fluorescent protein (GFP) expression in Müller cells in the 24
murine model of oxygen-induced retinopathy (OIR). This study compares the efficacy of 25
regulated and unregulated Müller cell delivery of endostatin in preventing 26
neovascularization in the OIR model. 27
Methods: Endostatin cDNA was cloned into plasmids with hypoxia regulated GFAP or 28
unregulated GFAP promoters, and packaged into self complementary Adeno 29
Associated virus serotype 2 vectors (scAAV2). Before placement in hyperoxia on 30
Postnatal day (P) 7, mice were given intravitreal injections of regulated or unregulated 31
scAAV2, capsid, or PBS. On P17, five days after return to room air, neovascular and 32
avascular areas, as well as expression of the transgene and vascular endothelial growth 33
factor (VEGF), were compared in OIR animals treated with a vector, capsid, or PBS. 34
Results: The hypoxia-regulated, glial-specific vector expressing endostatin reduced 35
neovascularization by 93% and reduced the central vaso-obliteration area by 90%, 36
matching the results with the unregulated GFAP-Endo vector. Retinas treated with the 37
regulated endostatin vector expressed substantial amounts of endostatin protein, and 38
significantly reduced VEGF protein. Endostatin production from the regulated vector 39
was undetectable in retinas with undamaged vasculature. 40
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Conclusion: These findings suggest that the hypoxia-regulated, glial cell-specific vector 41
expressing endostatin may be useful for treatment of neovascularization in proliferative 42
diabetic retinopathy. 43
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INTRODUCTION 58
Neovascularization in age-related macular degeneration and diabetic retinopathy 59
remains a major cause of vision loss and legal blindness in the world today. 60
Reduction, occlusion or loss of retinal or choroidal vessels are recognized as risk 61
factors for retinal, vitreal or choroidal neovascularization in diabetic retinopathy, 62
retinopathy of prematurity, and age-related macular degeneration. The consequent 63
retinal hypoxia increases levels of HIF-1, the master regulator of angiogenesis, which 64
regulates expression of many target genes, including at least thirty-one proangiogenic 65
factors and nine anti-angiogenic factors.1 66
The major growth factor associated with both physiological and pathological 67
angiogenesis is Vascular Endothelial Growth Factor (VEGF), expressed by several 68
ocular cell types, including Müller cells, RPE cells, pericytes, vascular endothelial cells, 69
and ganglion cells.2–5 VEGF mediates multiple events in an angiogenic program 70
characterized by increased vascular permeability (causing macular edema), 71
recruitment, proliferation, migration, adhesion, and organization of endothelial cells to 72
form tubular new vessels. The critical role of VEGF in many forms of ocular 73
neovascularization made it an attractive target for development of VEGF-targeting 74
therapies.6–8 The introduction of successful anti-VEGF treatments has dramatically 75
reduced vision loss in diabetic macular edema9,10 and neovascular forms of age-related 76
macular degeneration.11,12 However, better therapies are needed: the requirements for 77
multiple injections and frequent office visits place a burden on patients, 78
neovascularization may reappear when the treatment is stopped, and the injections are 79
associated with a low risk for elevated intraocular pressure, uveitis, vascular occlusion, 80
5
vitreous hemorrhage or retinal detachment.13–15 Moreover, there is wide variation in 81
patient responses to treatment; robust gain in vision is observed in about 30% of 82
patients, and 10% of patients do not respond to anti-VEGF treatments.8,16–18 A recent 83
large study (N=835 patients) found no link between SNPs in VEGF receptors, and 84
patient responses19 suggesting that continuous suppression of the VEGF signaling may 85
be more efficacious than monthly injections, and/or that other pathways are contributing 86
to the angiogenic responses to ocular hypoxia. 87
Gene therapy offers the advantage of local and sustained delivery of anti-88
angiogenic molecules, and has proven to efficiently suppress neovascularization in 89
animal models.20–24 Restricting expression of anti-VEGF molecules to periods when the 90
disease process is active should reduce the potential for complications associated with 91
prolonged reduction in VEGF, an important survival factor for photoreceptors, 92
endothelial cells, ganglion cells25, RPE and the ciliary process. VEGF inhibition has 93
been associated with thinning of the outer nuclear layer (ONL) in patients26; vessel 94
occlusion in animals and patients27,28 and in animals, with decreased ERGs29, reduced 95
thickness of the inner and outer nuclear layers30 and damage to RPE31 . (Despite these 96
observations, the overall safety record of intravitreal anti-VEGF drugs has been good; 97
the changes reported may be too subtle for routine clinical observation.) 98
Since hypoxia is a common feature of pathological NV leading to vision loss, 99
physiologically regulated gene delivery by low oxygen levels offers great potential for 100
more controlled gene therapy for ischemic ocular diseases. In order to target genes to 101
hypoxic cells, several hypoxia-sensitive gene switches have been developed, all of 102
which are based on hypoxia responsive elements (HRE).32–38 103
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The HREs are targeted by hypoxia inducible factor—a heterodimer of HIF-1α and HIF-104
1β. In normoxia, HIF-1α is hydroxylated and rapidly degraded by the proteasome. In 105
hypoxic conditions, HIF1α is stabilized and accumulates in the cytoplasm to form HIF1 106
dimers that bind the HREs in target genes. Incorporating HREs in our gene therapy 107
make it HIF-1 regulated, so the therapy will be activated only in retinal regions 108
experiencing hypoxia, or other pathological conditions activating HIF-1 regulated 109
angiogenesis. 110
We chose to target Müller cells, the major retinal glial cell, since it traverses the 111
retina, and would therefore experience hypoxic stress following capillary loss in 112
diabetes39,40 and expresses VEGF in oxygen-induced retinopathy.3 It is known that 113
Müller cell derived VEGF is a significant contributor to NV41 in the retina.42 Gene 114
therapy targeting Müller cells is an important addition to existing gene therapies that 115
target RPE cells; together they provide a means to selectively deliver antiangiogenic 116
therapy to either inner retina or outer retina/choroid. 117
To suppress ocular neovascularization, we selected endostatin for its profound 118
effects on angiogenesis affecting more than VEGF-dependent pathways. A cleavage 119
product of Collagen XVIII it inhibits endothelial cell proliferation, migration and survival 120
43, and it increases expression of anti-angiogenic factors (e.g. thrombospondin), and 121
inhibits levels of pro-angiogenic factors (e.g. HIF1a, ephrins, Ids)44. Endostatin blocks 122
VEGF-induced microvascular permeability by rapidly stabilizing occludin and tight 123
junctions in vessels.45 Following internalization into endothelial cells, either through 124
endocytosis or through clathrin-coated pits46, endostatin localizes to the nucleus47 and 125
7
reduces expression of VEGF and increases the expression of PEDF.48 The C-terminal 126
peptide of endostatin competes with VEGF for binding to VEGFR3.49 127
We previously reported that a promoter containing a hypoxia-responsive domain 128
together with a GFAP-cell specific promoter maintained cell-specificity and was hypoxia-129
inducible both in vitro and in vivo.38 The current study tested the hypothesis that our 130
hypoxia-regulated, glial cell specific AAV vector expressing endostatin would be as 131
effective in reducing neovascularization in the OIR model as gene therapy with 132
constitutively expressed endostatin.20,50 133
METHODS: 134
Construction of Plasmids and generation of scAAV2. 135
Previously tested promoter cassettes38 having the GFAP promoter or the REG- 136
GFAP (hypoxia regulated GFAP) promoter were cloned into the scAAV plasmid. The 137
human GFAP promoter (GfaABC1D domain of GFAP promoter, 681bp size), a gift from 138
Dr. Michael Brenner, University of Alabama, is a truncated promoter with subfragments 139
which in combination were previously shown to drive astrocyte specific gene expression 140
in transgenic mice.51 To make this promoter hypoxia responsive, we incorporated two domains 141
immediately upstream of the 5’ end of GFAP promoter: firstly, positioned most 5 prime, we 142
inserted a silencing region (122bp) (HRSE; provided by Keith Webster, University of Miami 143
Miller School of Medicine) and secondly, between the HRSE and the GFAP promoter we 144
inserted six copies of the previously described hypoxia response element (6XHRE; total size 145
108bp) of the phosphoglycerate kinase gene35
.To make endostatin encoding vectors, the 146
endostatin open reading frame of 612bp size (NCBI id NM_030582.3) without the stop 147
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codon was amplified from a commercially available plasmid (Catalog # puno1-hendo18, 148
Invivogen, San Diego, CA) by PCR using following primers (forward primer; 5’-149
GACaaccggtATGTACAGGATGCAACTC-3’ and reverse primer; 5- 150
GTATacgcgtCTACTTGGAGGCAGTCATG-3’ ) and cloned into self-complementary 151
Adeno-Associated Virus (scAAV) plasmid. To detect exogeneous endostatin, a FLAG 152
epitope sequence with appropriate restriction sites was synthesized (IDT DNA 153
Technology, Coralville, IA) and cloned into the 3’ end of endostatin (Fig. 1). The new 154
constructs were named as GFAP-Endo (scAAV-GFAP-Endostatin-FLAG) and REG-155
Endo (scAAV-HRSE-6XHRE-GFAP-Endostatin-FLAG). ScAAV serotype-2 viruses 156
were produced at the Gene Therapy Vector Core, University of North Carolina (Chapel 157
Hill, NC) and titers were determined by standard dot blot analysis. 158
Intravitreal Injection and Oxygen Induced retinopathy (OIR) model 159
All procedures involving mice were performed in accordance with the Association 160
for Research in Vision and Ophthalmology (ARVO) statement for the use of animals in 161
ophthalmic research and approved by the Institutional Animal Care and Use Committee 162
at Florida Atlantic University. The murine OIR model was used to compare the efficacy 163
of the unregulated GFAP-Endo and the hypoxia-regulated REG-Endo. Briefly, forty 164
pups age P7 (C57 Bl/6J mouse strain) were anesthetized with ketamine/xylazine and 165
given intravitreal injections of 1µl (2x109 viral particles) of either GFAP-Endo or REG-166
Endo (Fig. 1) in one eye and phosphate buffered saline or the empty AAV2 capsid 167
vector in the contralateral eye. P7 pups with their mothers were then either maintained 168
in normal room air, (room air controls 21% oxygen) or exposed to 75% O2 for 5 days in 169
a chamber, (OIR animals). At P12 pups were moved to room air conditions for 5 days. 170
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Exposure of neonatal mice to high oxygen (P7-P12) causes central vaso-obliteration by 171
P12; the reduced retinal vessels are insufficient in room air and the consequent 172
physiologic hypoxia induces peripheral neovascularization over the period P12-P17. All 173
the neonatal animals used for this study developed neovascularization. Following 174
ketamine/xylazine euthanasia at P17, eyes were rapidly collected for preparation of 175
flatmounts or for analysis of endostatin and protein expression. 176
Flat Mounts 177
Following removal of the cornea and lens, the eye cups were fixed with 4% 178
paraformaldehyde for an hour and washed in PBS. The retina was isolated from the eye 179
cup and stained with Isolectin B4-594 (Alexa Fluor 594 – I21413, Life Technologies, 180
Grand Island , NY, USA) at room temperature overnight. The retina was divided into 181
quadrants, which were flattened on a slide with ganglion cells down, cover-slipped with 182
Floromount G (Qiagen, Valencia, CA, USA ) and imaged using a Nikon inverted 183
fluorescence microscope. Flat mount techniques were successfully practiced in room air 184
control animals. 185
Analysis of vaso-obliteration and neovascularization 186
The percentage of vaso-obliteration was calculated by comparing the central 187
avascular area to total retinal area using Photoshop software.52,53 To quantify 188
peripheral neovascularization, the outer 2/3rd of each retinal quadrant was selected. 189
Using lasso tool the peripheral vascular area was selected and neovascular tufts were 190
quantified using magic tool as described by Connor et al (2009).52 The ratio of 191
peripheral neovascular area to the total vascular area was measured and expressed as 192
10
percentage. For each retina, neovascular areas in each quadrants were averaged to 193
represent that retina. 194
Western Blotting & ELISA 195
The retina was collected in radioimmunoprecipitation assay (RIPA) buffer with 196
protease inhibitors, homogenized on ice, and centrifuged. For western blots, 197
supernatant proteins (20 microg/well) were electrophoresed on a 12% Tris Glycine gel, 198
transferred to nitrocellulose, blocked for an hour with 5% dried milk in Tris-buffered 199
saline and incubated overnight with rabbit anti-FLAG primary antibody (Sigma, St. 200
Louis, MO, USA). Β-actin antibody was used as loading control for each sample. 201
Endostatin bands were detected by enhanced chemiluminescence using horse-radish 202
peroxidase conjugated secondary antibodies, and following the manufacturer’s 203
instructions (Pierce, Thermo Fisher Scientific, Rockford, IL, USA). 204
Endostatin and VEGF concentrations in retinal lysates were measured using the 205
RayBio® human endostatin and the RayBio® Mouse VEGF ELISA Kits (Ray Biotech 206
Inc.,Norcross, GA, USA) according to the recommended protocol. The concentration of 207
endostatin expressed by either GFAP-Endo or REG-Endo vector was normalized to that 208
in the PBS treated eyes. VEGF was normalized to the amount of protein in each 209
sample. Each experiment was performed in duplicate (n = 2), and each data point was 210
performed in triplicate. 211
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Statistical Analysis 212
Data are expressed as mean +/- SEM. Significance was determined using 213
analysis of variance with Newman-Keuls Multiple comparison test). Differences 214
between conditions were regarded as significant if p<0.05. 215
RESULTS: 216
Reduction in peripheral neovascular and central avascular areas: 217
Robust neovascularization was observed in both capsid and PBS treated eyes 218
(Fig. 2) from oxygen exposed mice. In comparison, the angiostatic effects of both 219
endostatin expressing vectors were clearly visible in flat mounted retinas (Compare A 220
and B in Figs. 2 and 3, respectively). Eyes treated with either GFAP-Endo or REG-Endo 221
had neovascular areas that were 90% lower (P< 0.001 for both) than those in PBS or 222
capsid injected eyes (Fig. 4). Neovascular areas in PBS and Capsid-injected eyes were 223
not significantly different. 224
Comparison of the vector-treated eyes (Figure 2 and 3; A and C) with their 225
contralateral control eyes treated with capsid or PBS (Figure 2 and 3; B and D) reveals 226
the dramatic reduction in central avascular area. The mean avascular areas in the 227
PBS and capsid treated eyes were almost 10-fold larger than in eyes treated with 228
GFAP-Endo or REG-Endo (P< 0.001; Fig. 5). 229
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Vector-Driven Endostatin Expression 230
Western blots clearly illustrate that exogenous endostatin levels achieved in the OIR 231
retinas were greater in the GFAP-Endo treated eyes than in the REG-Endo eyes. As 232
expected, the flagged endostatin was barely detectable in REG-Endo treated eyes of 233
animals maintained in room air (Fig. 6). ELISA of the flagged human endostatin 234
revealed 13-fold greater expression of endostatin by REG-Endo in OIR retinas than in 235
room air retinas ( 2.7 ± 0.8 ng/mg versus 0.2 ± 0.3ng/mg, respectively; P<0.01) (Fig. 7). 236
In room air mice, endostatin levels were 10-fold higher in GFAP-Endo treated retinas 237
than in REG-Endo treated retinas (2.3 ± 0.4 ng/mg versus 0.2 ± 0.3ng/mg, respectively; 238
P< 0.01 (Fig. 7). The 13-fold difference in endostatin expression by REG-Endo in room 239
air and in OIR retinas is comparable to the 12-fold difference observed in our in vitro 240
experiments (Fig. 4 in our previous manuscript38). Endostatin expression in GFAP-241
Endo injected eyes was not significantly different in OIR animals by comparison to room 242
air animals. 243
Reduction of VEGF levels in OIR retina 244
VEGF protein levels in P17 control retinas (PBS injected eyes) exposed to high 245
oxygen were significantly higher than those in room air animals (365 ± 20 pg/mg versus 246
85 ± 23 pg/mg; P<0.001) (Fig. 8). Intravitreal injection of either GFAP-Endo (144± 28 247
pg/mg) or REG- Endo (153 ± 38 pg/mg) significantly reduced VEGF levels (P< 0.001; 248
Fig. 8) compared to PBS injected eyes. No significant difference was observed between 249
VEGF levels in eyes treated with GFAP-Endo or REG- Endo vectors. 250
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DISCUSSION 251
Therapeutic Efficacy 252
These data represent the first demonstration of the efficacy of hypoxia-regulated, 253
cell-specific anti-angiogenic gene therapy for the retina. In the OIR model, our new 254
REG-Endo vector reduced neovascular area by 93% and the central avascular area by 255
90%. These results are comparable to those achieved with the constitutively expressed 256
GFAP-Endo, and with reports that the number of pre-retinal endothelial cell nuclei was 257
reduced by 86% by AAV-mediated endostatin50 and by 75% by an adenoviral 258
endostatin vector.54 259
The Müller cell specificity of our promoter has important implications because this 260
major glial cell traverses the width of the retina and may experience a distinct pattern of 261
hypoxia. While our glial cell specific and hypoxia regulated vectors will express 262
endostatin selectively in Müller cells, questions remain regarding the effects of the 263
vectors in respect to the location of hypoxia within the retina that may trigger gene 264
expression. For example a specific defined band of hypoxia in a region of the retina may 265
result in transgene expression from Müller cells that traverse this region but one 266
constraint may be the magnitude of cell surface for individual Müller cells that 267
experience oxygen deprivation. For a particular Müller cell, once the transgene is 268
induced it is likely that the endostatin protein product would be secreted from all 269
locations on the Müller cell membrane and therefore at a wide range of depths within 270
the retina. 271
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We have demonstrated regulated expression from our REG-Endo vector both in 272
terms of increased protein product (as shown in ELISA at P17 by comparison to 273
expression in room air) and in terms of functional outcome (inhibition of 274
neovascularization). From the literature and our own data, it is clear that extensive 275
inhibition of neovascularization is obtained with AAV-CMV-Endostatin50as well as from 276
our own GFAP-Endo and REG-Endo vectors. Importantly the mechanisms of 277
endostatin action are poorly understood and include inhibition of VEGF induced 278
permeability, actions in the nucleus, decreased VEGF signaling and/or production and 279
increased PEDF expression. Hence the production of endostatin ubiquitously under the 280
CMV promoter may result in differences in endostatin action which occur in a very 281
different and progressively changing multi-cellular microenvironment from that obtained 282
with a Müller restricted vector or from REG-Endo. A novel feature of our vectors is 283
secretion of endostatin specifically from Müller cells or from hypoxic Müller cells eliciting 284
successful inhibition of neovascularization. Such inhibition of neovascularization by 285
endostatin has not been elicited previously from either Müller cell-specific or combined 286
Müller cell- specific/hypoxia regulated vectors. While the exact details of endostatin 287
action are only partially understood, it is likely that eliciting the more selective patterns 288
of endostatin distribution and release will result in distinct patterns of vascular inhibition 289
compared to the more diffuse ubiquitous patterns of multicellular secretion obtained with 290
AAV-CMV-endostatin. Furthermore the REG-Endo vector will result in activated therapy 291
that is disease specific and corresponds to emergence of a hypoxic microenvironment 292
instead of continuous and unvarying rates of production initiating soon after vector 293
delivery. (Note: The activation of our promoter is likely to differ in astrocytes that reside 294
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in the inner layers of the retina and therefore may experience different levels of hypoxia 295
compared to cells. As a result, the responses in these two cell types could be different 296
both in timing and in levels of activation.) 297
It has been suggested Müller that sustained delivery of a transgene may lead to 298
tissue toxicity over time23 ; as discussed earlier, reduced VEGF has been linked to 299
retinal damage in animals. If the transgene is regulated in response to the pathologic 300
state of surrounding tissue, it can be effective during the period of the disease and 301
turned off once the disease progression is stabilized. If the patient has a recurrence of 302
the pathology, the expression will turn on again. Although our vector successfully 303
inhibited neovascularization within a short period of time, the effectiveness in ensuring 304
more long-term transgene expression when necessary requires further investigation. 305
Two major advantages of this strategy are that the anti-angiogenic therapy is 306
delivered as vessels start to form, when they are most amenable to treatment, and that 307
there is a high therapeutic index because therapy is delivered only when and where 308
needed; the remaining healthy retina would be exposed to a much lower dose. and only 309
for the duration needed, as expression is suppressed in normoxic tissue. 310
Retinal Endostatin Levels 311
Our regulated vector produced approximately 2-3 ng of endostatin/mg of retinal 312
homogenate (Fig. 7), which corresponds closely to the 1-2 ng of endostatin reported 313
previously with the constitutive VMD2-RPE specific promoter.21 While these levels are 314
less than 10-14 ng of endostatin delivered by a CMV promoter, it is likely that high 315
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absolute levels of anti-angiogenic protein are not necessary to achieve a substantial 316
therapeutic effect. The key difference between REG-Endo and CMV-endostatin is that 317
for REG-Endo, levels of endostatin produced will be more than 10-fold lower in 318
normoxic retina than in OIR animals. 319
GFAP activity and endostatin delivery. 320
Endostatin levels were higher in GFAP-Endo treated eyes from OIR mice than in 321
those from similarly treated room air mice (Fig.7). Müller cells become reactive and 322
induce GFAP in response to most types of retinal injury. Reactive gliosis is a hallmark 323
of the retinal response in the OIR model.55 As a result, the basal level of the unregulated 324
GFAP-Endo will increase as GFAP expression increases, and will remain active during 325
retinal inflammation and damage. In contrast, REG-Endo will be constitutively silent, 326
activated in hypoxia and shut-off by declining HIF-1 levels, at least by P26, when the 327
OIR retina has “resorbed” the newly formed vessels.2 Once activated by hypoxia, the 328
increase in GFAP activity that is associated with the reactive phenotype in Müller cells 329
will further boost the production of endostatin. 330
Change in VEGF levels 331
Like others, we found that OIR retinas had significantly higher VEGF than room 332
air retinas. The amount of VEGF produced in the OIR retinas was comparable with that 333
in other studies.56 VEGF levels at P17 were approximately 60% lower in the endostatin 334
eyes. The significant reduction in central avascular area would certainly contribute to a 335
decline in VEGF. It is interesting that the reduction in VEGF in these experiments is 336
17
comparable to that observed in eyes injected with 500 times higher (1 µg) dose of 337
endostatin on P12 in the OIR model.57 This might indicate that the REG-Endo produces 338
endostatin at close to saturating levels. 339
Hypoxia-regulated gene therapy 340
Hypoxia-regulated gene therapy was introduced by Prentice et al (1997) as a 341
means to deliver cardioprotective genes to myocardium experiencing transient or 342
intermittent ischemia.36 The field has grown slowly since that time with reports on 343
hypoxia-regulated gene therapy targeting hypoxic regions of tumors, followed by reports 344
on cardiac ischemia and/or ischemia reperfusion injury to heart or neural tissue. 345
Bainbridge et al first reported hypoxia-regulated expression of a reporter gene in the 346
retina; it was neither cell-specific nor suppressed in normoxic retina32. Our group 347
previously reported a retinal pigment epithelium (RPE)-specific AAV vector that was 348
robustly responsive to hypoxia and silenced in normoxia35 as a potential platform for 349
treatment of choroidal neovascularization. To develop a platform targeting intraretinal 350
neovascularization, a new hypoxia-responsive, glial-specific AAV vector was 351
constructed to target Müller cells, and its specificity and responsiveness were verified in 352
vitro, and in the OIR model of retinal neovascularization.38 353
The highly sensitive response of the REG-Endo vector to hypoxia results from its 354
design. We utilized hypoxia response elements which are consensus DNA binding sites 355
for the HIF-1 transcription factor, which is kept at low levels in normoxia by the oxygen-356
dependent degradation of the HIF-1alpha subunit. Hypoxia regulated vectors have 357
been of value in different model systems for expressing a range of factors including pro-358
18
survival components, angiogenic factors and growth factors.58–61 By incorporating 359
multimers of the HRE, we increased the response of the promoter to hypoxic conditions; 360
the neuronal silencing element further ensures very low basal expression in normoxia 361
prior to hypoxic induction. REG-ENDO was made cell specific by virtue of the 681 bp 362
hybrid regulatory domain from the GFAP gene (See Methods section). 363
HRE-regulated switch in normoxia 364
A key question regarding timing of transgene expression will be whether 365
endostatin expression ceases once the pathology has subsided. Cell specific and 366
hypoxia-regulated vectors have been shown to switch off in other systems including 367
heart 36,62and cancer.63,64 Given the tight correspondence between tissue hypoxia and 368
the induction, maintenance and termination of HIF signaling, it is anticipated that the 369
activity of REG-Endo will closely correlate with retinal hypoxia. 370
Implications for the future. 371
This new strategy for anti-angiogenic gene therapy opens an avenue of 372
prophylactic gene therapy for patients at high risk for neovascularization. Theoretically, 373
high risk patients could be treated with a gene therapy that would only be activated 374
when pathological conditions begin to stimulate neovascularization. Therapy would 375
initiate before the patient experienced any symptoms or loss of vision. 376
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SUMMARY 379
The major findings of the present study were the use of a hypoxia-regulated, 380
retinal glial cell specific promoter driving the expression of the human endostatin gene 381
to dramatically reduce peripheral neovascularization and central in the OIR mouse 382
model of neovascularization. Our results demonstrated the effect of exogenous 383
endostatin on VEGF expression levels in retina, and confirmed that the regulated vector 384
produced sufficient endostatin to inhibit hypoxia-induced neovascularization in the 385
murine OIR model. These results suggest that the localized production of endostatin in 386
ischemic retina reduces pathological angiogenesis and promotes physiological 387
angiogenesis. 388
ACKNOWLEDGMENTS 389
The authors would like to thank Sanghamitra Das for providing technical assistance on 390
this project. The authors would like to acknowledge Dr. Michael Brenner (University of 391
Alabama) for contributing the plasmid containing the GFAP promoter. Dr. George 392
(Tyler) Smith offered helpful suggestions in the design of the experiments. 393
394
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Figure legends: 592
Figure 1: Schematic diagram of vectors used in this study. 593
The promoter elements and endostatin cDNA are incorporated into self-complementary 594
Adeno Associated Vectors (ScAAV2). (A) GFAP-Endo vector; this vector has human 595
glial fibrillary acidic protein (GFAP) promoter (681bp) and human endostatin (Endo) 596
cDNA open reading frame (612bp) fused with FLAG epitope . (B) REG-Endo vector; 597
this vector includes hypoxia regulated sequence (HRSE-6XHRE, 230bp) along with 598
GFAP promoter and endostatin cDNA fused with FLAG epitope. SV40 poly A (SV40pA) 599
sequence and inverted tandem repeat sequences (ITR) are present in scAAV2 vectors. 600
(dITR denoted the mutated ITR in 5’ end). Construction of above vectors is described in 601
methods section. 602
Figure 2: Representative micrographs of flat mounted P17 retinas treated with 603
GFAP-Endo. 604
Light micrographs of retinas from mice exposed to high oxygen were isolated, retinal 605
whole mounts made, and blood vessels identified with lectin staining. In response to 606
high oxygen followed by room air, the central retina loses much of the normal 607
vasculature whereas neovascularization occurs in the peripheral retina indicated by the 608
formation of neovascular tufts. Retina from GFAP-Endo treated eye (A) shows 609
reduction of both the central avascular area (solid white square) and peripheral 610
neovascular area (square with dashed white lines) compared to contralateral capsid 611
injected eye (B). Magnified view of the peripheral retina shows reduced neovascular 612
30
tufts in GFAP- Endo treated eye (arrows, C) compared to capsid injected eye (arrows, 613
D). Magnified view of central avascular area shows reduction of vaso-obliteration and 614
increased vascularity in GFAP- Endo treated eye (arrows, E) compared to capsid 615
injected eyes (arrows, F). Note, the “hot spot” in the retina (asterisk, A) is an artifact 616
due to accidental puncture during the injection. (Scale bar; A and B = 1000um; C, D, E, 617
and F = 100um) 618
Figure 3: Representative micrographs of flat mounted P17 retinas from eyes 619
treated with REG-Endo. 620
Light micrograph of retinal whole mount from REG-Endo treated eye (A) shows 621
reduction of both central avascular area (white square) and peripheral neovascular area 622
(square with dashed white lines) compared to contralateral PBS injected eye (B). 623
Magnified view of peripheral neovascular retina shows reduced neovascular area in 624
REG- Endo treated eye (arrows, c) compared to PBS injected eye (arrows, D). 625
Enlarged view of central avascular area shows amelioration of vaso-obliteration and 626
normal vascular patterning in REG- Endo treated eye (arrows, E) compared to 627
contralateral PBS injected eye (F). The bright strands in (e) and (f) are remnants of 628
hyloid vessels. (Scale bar; a and b = 1000um; c, d, e, and f = 100um) 629
Figure 4: Reduction of peripheral neovascularization. 630
A) Mean neovascular areas (Mean ± S. E) in eyes treated with GFAP or REG-631
endostatin vector, PBS, or Capsid . Eyes treated with either GFAP-Endo or REG-Endo 632
had neovascular areas (2.2± 0.5% and 1.6± 0.6%, respectively) that were 90% lower 633
31
than eyes injected with either PBS or capsid injected eyes (*P<0.001). Neovascular 634
areas in PBS (n=11) and Capsid-injected eyes (n=10) were not significantly different 635
(24.8 ± 3.2 %, and 22.8 ± 4.1%, respectively) (P < 0.056 ). B) Paired comparison of 636
neovascular area in eyes from mice injected with endostatin vector in one eye and 637
Capsid or PBS in the contralateral eye. Treatments and mouse number are indicated 638
under each pair of bars. 639
Figure 5: Reduction of central avascular area. 640
A) Central avascular area (Mean ± S. E) in retinas treated with GFAP or REG-641
Endostatin vector, PBS, or capsid. Significant reduction of the central avascular area 642
was observed in GFAP-Endo (2.34 ± 1.4 %, n =9) and REG-Endo (2.31 ± 1.6 %, n = 9) 643
injected eyes compared to either PBS (21.1 ± 1.9 %, n =11) or Capsid (21.87 ± 2.5 %, n 644
= 10) injected eyes (*P<0.001). There was no significant difference in the central 645
avascular area in eyes injected with either PBS or Capsid. (* P < 0.02 ).B) Paired 646
comparison of avascular areas in eyes injected with endostatin vector in one eye and 647
either capsid or PBS in the contralateral eye. (n = Number of individual mice). 648
Figure 6: FLAG tagged endostatin in mouse retina at P17: 649
Lane 1 shows that eyes treated with REG-Endo vector resulted in barely detectable 650
expression of endostatin in room air. Lane 2 and Lane 3 demonstrate expression of 651
endostatin (22 kDa) by REG-Endo vectors was greater in OIR retinas but still less than 652
the expression from the GFAP-Endo vector. 653
32
Figure 7: Expression of endostatin by regulated and unregulated vectors in room 654
air or OIR retinas. 655
ELISA analysis of retinal endostatin expression in mice that were injected with GFAP-656
Endo or REG-Endo, then placed in oxygen for the OIR model or raised in room air. In 657
the room air groups, retinal endostatin concentration in the REG-Endo mice was only 658
10% of that in the GFAP-Endo mice. Endostatin concentration in the retinas of the 659
REG-Endo mice in the OIR model was 12-fold greater than in REG-Endo mice in room 660
air. Retinal endostatin expression in the OIR model was statistically equivalent in the 661
REG-Endo and GFAP-Endo mice. Groups with different letters differ significantly 662
(P<0.01 for all comparisons; N=6 in all groups). 663
Figure 8: VEGF levels measured by ELISA at P17. 664
VEGF levels were higher in retinas from PBS treated OIR mice than in room air 665
controls. Treatment with either GFAP-Endo or REG-Endo reduced VEGF levels in the 666
OIR retinas. ( * P < 0.001) 667