Inhibition of Choroidal Neovascularization via Brief Subretinal Exposure to a Newly Developed Lentiviral Vector Pseudotyped with Sendai Viral Envelope Proteins

Abstract

Lentiviral vectors are promising tools for the treatment of chronic retinal diseases, including age-related macular degeneration (AMD), as they enable stable transgene expression. On the other hand, Sendai virus (SeV) vectors provide the unique advantage of rapid gene transfer. Here we show that novel simian immunodeficiency viral vectors pseudotyped with SeV envelope proteins (SeV-F/HN-SIV) achieved rapid, efficient, and long-lasting gene transfer in the mouse retina. Subretinal exposure to SeV-F/HN-SIV vectors for only a few minutes resulted in high-level gene transfer to the retinal pigment epithelium, whereas several hours were required for gene transfer by standard vesicular stomatitis virus G-pseudotyped SIV vectors. Transgene expression continued over a 1-year period. SeV-F/HN-SIV vector-mediated retinal overexpression of soluble Fms-like tyrosine kinase-1 (sFlt-1) or pigment epithelium-derived factor (PEDF) significantly suppressed laser-induced choroidal neovascularization (CNV). Histologically, 6-month-long sustained overexpression of PEDF did not adversely affect the retina; however, that with sFlt-1 resulted in photoreceptor degeneration associated with choroidal circulation defects. These data demonstrate that brief subretinal administration of SeV-F/HN-SIV vectors may facilitate safe and efficient retinal gene transfer, and suggest the therapeutic potential of PEDF with a higher safety profile for treating CNV in AMD patients.

Introduction

T he development of choroidal neovascularization (CNV) is a principal pathological feature of age-related macular degeneration (AMD), the leading cause of adult blindness in industrialized countries (Lopez et al., 1991; Friedman et al., 2004; Miyazaki et al., 2005). One of the major factors that induce CNV is vascular endothelial growth factor-A (VEGF-A), a diffusible cytokine that promotes angiogenesis and vascular permeability (Ishibashi et al., 1997; Krzystolik et al., 2002). Clinical data analyses have revealed that the intravitreal administration of VEGF-A antagonists such as ranibizumab, bevacizumab, and pegaptanib arrests CNV progression and leakage from CNV, and improves visual acuity (Gragoudas et al., 2004; Avery et al., 2006; Brown et al., 2006; Rosenfeld et al., 2006). However, these drugs need to be used repeatedly at 4- to 6-week intervals, which raises concerns about injection-related adverse events including ocular inflammation, retinal injury, and endophthalmitis.

Advances in gene therapy technologies have been introduced as components of novel therapeutic approaches to the treatment of retinal diseases. Among several vector systems, recombinant adeno-associated viral (rAAV) and lentiviral vectors are likely to be useful for patients with chronic progressive retinal diseases, as these vectors can achieve gene transfer in nondividing cells and yield long-term transgene expression (Miyoshi et al., 1997; Acland et al., 2001; Lai et al., 2002; Ikeda et al., 2003). Clinical studies demonstrated the safety and efficacy of the subretinal delivery of rAAV carrying the RPE65 gene for patients with Leber's congenital amaurosis, thereby providing the proof of concept for retinal gene therapy strategies (Bainbridge et al., 2008; Hauswirth et al., 2008; Maguire et al., 2008; Cideciyan et al., 2009). The clinical study has shown that the viral vector solution left in the subretinal space is almost fully absorbed by 24 hr after injection (Bainbridge et al., 2008); however, it is more preferable to remove the vector solution and resolve the retinal detachment (RD) during gene transfer surgery, because the outer retinal cells are deprived of trophic and metabolic support from the retinal pigment epithelium (RPE) and choroid vessels during RD. Hauswirth and colleagues reported that retinal thinning was observed in one of three patients after subretinal injection of rAAV-RPE65 (Hauswirth et al., 2008). We developed novel simian lentiviral vectors pseudotyped with Sendai virus (SeV) hemagglutinin–neuraminidase (HN) and fusion (F) envelope proteins (SeV-F/HN-SIV) (Kobayashi et al., 2003). The F and HN proteins of SeV mediate viral attachment and penetration (Lamb and Kolakofsky, 1996), and SeV-derived vectors have shown efficient gene transfer with only a few minutes of vector–cell interaction (Yonemitsu et al., 2000; Masaki et al., 2001). Therefore, SeV-F/HN-SIV vectors are expected to facilitate rapid transfection, and they may enable the removal of the vector solution shortly after injection.

Neovascularization is thought to be regulated by the balance between angiogenesis inducers and inhibitors (Folkman, 1995). Soluble Fms-like tyrosine kinase-1 (sFlt-1)/VEGF receptor (VEGFR)-1, a splice variant of the VEGF receptor Flt-1, is an endogenous inhibitor of VEGF-A (Kendall et al., 1996), and has been used successfully to attenuate CNV in experimental models (Honda et al., 2000; Bainbridge et al., 2002; Gehlbach et al., 2003). However, studies have indicated that VEGF-A acts as a survival factor in normal vascular endothelial cells and neural cells (Darland et al., 2003; Lee et al., 2007; Maharaj et al., 2008), and some investigators have warned that a long-term blockade of VEGF signaling may lead to vascular and tissue dysfunction in the retina, as observed in the case of other systemic organs (Maynard et al., 2003; Levine et al., 2004; Hurwitz and Saini, 2006). Pigment epithelium-derived factor (PEDF) is a secreted glycoprotein isolated from the conditioned medium of human RPE (Tombran-Tink and Johnson, 1989; Steele et al., 1993), and it has been shown to exhibit both antiangiogenic and neuroprotective properties (Taniwaki et al., 1995; Dawson et al., 1999). Retinal gene transfer of PEDF by adenoviral vectors substantially inhibited experimental CNV by inducing apoptosis in activated endothelial cells (Mori et al., 2002); and this approach is currently under evaluation in a clinical trial investigating its efficacy in AMD patients (Campochiaro et al., 2006). However, the effects of long-term PEDF overexpression on the normal retinal vasculature and neurons remain unknown.

In the present study, we aimed to evaluate the availability of SeV-F/HN-SIV vectors for retinal gene transfer. We demonstrated the rapid and efficient transduction ability of these vectors, both in vitro and in vivo. Using this new vector system, we assessed the efficacy and safety of long-term overexpression of the antiangiogenic agents sFlt-1 and PEDF in a mouse model of laser-induced CNV.

Material and Methods

SIV vectors

To produce third-generation recombinant SIV-based lentiviral vectors, HEK 293T cells were transfected with the packaging vector; gene transfer vectors encoding enhanced green fluorescent protein (EGFP), hPEDF, or hsFlt-1 driven by the cytomegalovirus promoter; the ReV expression vector; and the envelope vector (vesicular stomatitis virus G glycoprotein [pVSV-G] [Clontech Laboratories, Mountain View, CA] or pSeV-F/HN with a truncation of the cytoplasmic tail of F and the addition of the cytoplasmic tail of the SIV transmembrane envelope protein to the N terminus of HN) (Kobayashi et al., 2003). The SIV vector lacking a transgene cassette (SeV-F/HN-SIV-Empty) was used as the control vector. The U3 region in the 3′ and 5′ long terminal repeat of SIV was deleted to induce self-inactivation. The viral titer was determined by the transduction of HEK 293T cells and expressed as transducing units (TU) per milliliter, and the viruses were kept at −80°C until just before use.

Cell culture and in vitro gene transfer

ARPE-19 cells, a human RPE-derived cell line, were purchased from the American Type Culture Collection (Manassas, VA). ARPE-19 cells were seeded in 24-well plates at 1 × 10⁵ cells per well in serum-free Dulbecco's modified Eagle's medium (DMEM)–F12. Twelve hours later, either SeV-F/HN-SIV-luciferase or VSV-G-SIV-luciferase was added to each well at a multiplicity of infection (MOI) of 10. After various times of incubation with each vector solution, the culture medium was removed, the cells were washed twice with phosphate-buffered saline (PBS), and fresh medium was added. At 48 hr after gene transfer, the cells were harvested and subjected to luciferase assay.

Animals and in vivo gene transfer

Adult C57BL/6 mice were maintained humanely, with proper institutional approval, and in accordance with the statement of the Association for Research in Vision and Ophthalmology. All animal experiments were carried out according to approved protocols and in accordance with the recommendations for the proper care and use of laboratory animals by the Committee for Animals, Recombinant DNA, and Infectious Pathogen Experiments at Kyushu University and according to the Law (No. 105) and Notification (No. 6) of the Japanese Government. The subretinal injection of each solution was performed as previously described (Murakami et al., 2008b). Briefly, mice were anesthetized by inhalation of ether. A 30-gauge needle was inserted into the subretinal space of the peripheral retina in the nasal hemisphere via an external transscleral transchoroidal approach. Subretinal injection of vector solution (SeV-F/HN-SIV or VSV-G-SIV vector; 2.5 × 10⁷ TU/ml × 2 μl) resulted in a dome-shaped detachment of about half the retina. To assess the effects of vector–cell interaction time on retinal gene transfer, we used two different subretinal administration techniques: either simple injection (leave) or removal 5 min after gene transfer by draining the subretinal vector solution and injecting balanced salt solution (BSS) after drainage (remove). The following assessments for duration of transgene expression, the efficacy of antiangiogenic factors for laser-induced CNV, and their retinal toxicity were performed by the remove procedure. Eyes that sustained marked surgical trauma (e.g., retinal or subretinal hemorrhage, bacterial infection) were excluded from further analyses.

Luciferase assay

Procedures used for the luciferase assay have been described previously (Ikeda et al., 2002). ARPE-19 cells and enucleated mouse eyes were treated with 1 × lysis buffer (Promega, Madison, WI) with a protease inhibitor cocktail and centrifuged, and then 20-μl samples of the supernatants were mixed with 100 μl of luciferase assay buffer. Light intensity was measured with a luminometer (model LB 9507; Berthold Technologies, Bad Wildbad, Germany) with 10-sec integration.

Detection of GFP expression in vivo and indocyanine green angiography

GFP expression in the mouse retina was examined with a scanning laser ophthalmoscope (Heidelberg retinal angiograph; Heidelberg Engineering, Heidelberg, Germany), using a 488-nm excitation laser light and a 500-nm barrier filter. Indocyanine green (ICG) angiography was performed 7 min after intraperitoneal injection of 0.2 ml of 10% ICG, using a 795-nm excitation diode laser light and an 810-nm barrier filter, as previously described (Janssen et al., 2008).

Histological examination

Mouse eyes were enucleated, and both paraffin and cryosections were prepared. To prepare the paraffin sections, the eyes were fixed with 4% paraformaldehyde in PBS for 24 hr, and were then mounted in paraffin. For the cryosections, the eyes were frozen in liquid nitrogen, and 5-μm-thick sections were prepared along the horizontal meridian. The sections were subsequently stained with hematoxylin and eosin. The number of cells in the outer nuclear layer was counted per 250 μm at six points around the retinal section (A1–A3, from the ora serrata to the optic nerve of the temporal hemisphere; A4–A6, from the optic nerve to the ora serrata of the nasal hemisphere).

Immunohistochemistry

Five-micrometer-thick cryosections were cut, air dried, and fixed in cold acetone for 10 min. The sections were blocked with 3% nonfat dried milk and labeled with rabbit anti-GFP polyclonal antibody (diluted 1:300; Invitrogen Molecular Probes, Eugene, OR) at 4°C for 24 hr. After biotinylated goat anti-rabbit IgG (H+L) (diluted 1:200; Vector Laboratories, Burlingame, CA) was applied as a secondary antibody, the cells were incubated with fluorescein isothiocyanate (FITC)-conjugated streptavidin (diluted 1:100; BD Biosciences, San Jose, CA). After labeling, 4′,6-diamidino-2-phenylindole (DAPI) was used to counterstain the nuclei. Immunofluorescence images were acquired with an Olympus BX51 microscope with a fluorescence attachment (Olympus, Tokyo, Japan). For negative controls, the primary antibody was omitted.

Enzyme-linked immunosorbent assay

The protein content of mouse eyes was determined with an enzyme-linked immunosorbent assay (ELISA) kit for human PEDF (not available for mouse PEDF; Chemicon International, Temecula, CA) and human sFlt-1 (not available for mouse sFlt-1; R&D Systems, Minneapolis, MN). For the preparation of ocular tissue, conjunctival and muscular tissues were removed from enucleated eyes. The eyes were washed with PBS, minced with scissors in 500 μl of 1 × lysis buffer with a protease inhibitor cocktail, and centrifuged at 15,000 rpm for 5 min at 4°C. The supernatants were subjected to ELISA according to the manufacturer's instructions.

Laser-induced CNV

Laser photocoagulation (630 nm, 150 mW, 0.1 sec, 75 μm) was performed on one eye (four spots per eye) of each animal, using a slit-lamp delivery system (NIDEK, Aichi, Japan), as previously described (Tsutsumi et al., 2003). Burns were performed at the 2, 5, 7, and 10 o'clock positions around the optic disk such that two laser-treated sites were within the vector-injected area, and the other two laser-treated sites were located outside of that area. Laser injury disrupted the RPE, Bruch's membrane, and choroid, and induced the subsequent proliferation and migration of choroidal endothelial cells, resulting in CNV. On day 14 after the laser induction of injury, the eyes were enucleated and fixed with 4% paraformaldehyde. The eyecups, obtained by removing the anterior segments and the entire neural retina, were incubated with FITC-conjugated isolectin B4 (Vector Laboratories) at 4°C for 24 hr. Then, CNV was visualized by Olympus BX51 fluorescence microscopy, and the area was measured with National Institutes of Health (Bethesda, MD) ImageJ software.

Transmission electron microscopy

The eyes were enucleated, and the posterior segments were fixed in 2.5% glutaraldehyde and 2% paraformaldehyde in 0.1 M cacodylate buffer with 0.08 M CaCl₂ at 4°C. Sections of retina, RPE, and choroid complex in the vector-injected area were postfixed for 1.5 hr in 2% aqueous OsO₄, dehydrated in ethanol and water, and embedded in Epon. Ultrathin sections were cut from blocks and stained with saturated, aqueous uranyl acetate and Sato's lead stain. The specimens were observed with a Hitachi H-7650 electron microscope (Hitachi High-Technologies, Tokyo, Japan).

Statistical analyses

All values are expressed as means ± SD. Statistical differences were assessed by analysis of variance (ANOVA) followed by Tukey–Kramer adjustments for multiple comparisons. Numbers per group are as indicated. A p value of less than 0.05 was considered statistically significant.

Results

SeV-F/HN-SIV vectors require only a brief vector–cell interaction to achieve efficient gene transfer

We previously demonstrated that recombinant Sendai viral (rSeV) vectors exhibited efficient gene transfer into the RPE of the rat retina with a brief exposure time (Ikeda et al., 2002). To determine whether SeV F- and HN-pseudotyped SIV vectors would be capable of rapid and efficient transfection, we first investigated the vector–cell interaction time-dependent transgene expression level. In a human RPE-derived cell line, ARPE-19, conventional VSV-G-pseudotyped SIV (VSV-G-SIV) vectors encoding luciferase showed an interaction time-dependent increase in luciferase expression, and more than 24 hr of exposure was required to achieve maximal gene transfer (Fig. 1A). In contrast, with SeV-F/HN-SIV-luciferase, only 1 min of exposure yielded efficient luciferase expression at levels similar to those observed with 48 hr of exposure (Fig. 1B). Moreover, the luciferase expression levels achieved with SeV-F/HN-SIV vectors were more than 10-fold higher than the peak value obtained with VSV-G-SIV vectors (Fig. 1A and B).

FIG. 1.

Effect of vector–cell interaction time on gene transfer to ARPE-19 cells. (A and B) ARPE-19 cells were exposed to (A) VSV-G-SIV-luciferase or (B) SeV-F/HN-SIV-luciferase at a multiplicity of infection (MOI) of 10 for each time period, and the cells were washed three times with PBS (n = 4 each). Forty-eight hours later, the cells were subjected to luciferase assay. VSV-G-SIV vectors required more than 24 hr of exposure time to achieve maximal gene transfer, whereas only 1 min of exposure was sufficient for SeV-F/HN-SIV vectors to reach the maximal expression level. (Note the log scale.) Cont., control; RLU, relative light units.

Next, we assessed the in vivo transduction efficiency of SeV-F/HN-SIV vectors in the mouse retina. To assess the effects of vector–cell interaction time on retinal gene transfer, we subretinally injected the SIV vectors by one of two different techniques, that is, either the vector solution was left in the subretinal space (leave), or the solution was removed 5 min after vector injection (remove). In eyes treated with VSV-G-SIV-luciferase, transgene expression in the remove group was markedly reduced, by approximately one-tenth, in comparison with that of the leave group (Fig. 2A). In contrast, eyes treated with SeV-F/HN-SIV-luciferase showed efficient transgene expression even in the remove group, and the luciferase expression levels in the SeV/F/HN-SIV-vector remove group were 1.3-fold higher than those of the VSV-G-SIV-vector leave group (Fig. 2A). These data indicate that brief vector–cell contact is sufficient for SeV-F/HN-SIV vectors to achieve efficient retinal gene transfer.

FIG. 2.

SeV-F/HN-SIV vector-mediated retinal gene transfer with brief exposure time. (A) Effect of vector exposure time in vivo. VSV-G-SIV-luciferase or SeV-F/HN-SIV-luciferase was injected into the subretinal space of mice by two different techniques: simple injection (leave), or removal 5 min after injection (remove). The eyes were subjected to luciferase assay 2 weeks after gene transfer (n = 5 or 6). (B) Retinal GFP expression in eyes treated with SeV-F/HN-SIV-EGFP. GFP fluorescence was assessed by scanning laser ophthalmoscopy 6 months (6M) and 1 year (1Y) after vector injection. ON, optic nerve. (C) Immunohistochemical staining for GFP in retina treated with SeV-F/HN-SIV-EGFP. Arrows indicate staining in the retinal pigment epithelium (RPE). DAPI, 4′,6-diamidino-2-phenylindole; INL, inner nuclear layer; ONL, outer nuclear layer. Scale bar, 100 μm.

Stable long-term transgene expression in RPE by SeV-F/HN-SIV vectors

Our previous study demonstrated that subretinal injection of VSV-G-SIV vector at 2.5 × 10⁷ TU/ml resulted in sustained transgene expression over a 1-year period in the rat retina (Ikeda et al., 2003). To determine the longevity of SeV-F/HN-SIV vector-mediated retinal gene transfer, we monitored the time course of transgene expression using GFP as a reporter. Mouse retinas treated with SeV-F/HN-SIV-EGFP by the remove procedure showed intense GFP fluorescence in an area corresponding to the vector-injected area, and the extent of GFP fluorescence was maintained for at least 1 year (Fig. 2B). Histological examination revealed that GFP expression was located in the RPE layer (Fig. 2C), as previously observed in the case of rSeV-mediated retinal gene transfer (Ikeda et al., 2002; Murakami et al., 2008b). Taken together, these findings indicate that SeV-F/HN-SIV vectors exhibit the advantageous features of both SeV vectors and SIV vectors, that is, rapid transduction and long-term transgene expression ability, in retinal tissue.

SeV-F/HN-SIV vector-mediated retinal delivery of antiangiogenic factors suppresses laser-induced CNV

Next, we sought to investigate the effects of SeV-F/HN-SIV vector-mediated PEDF or sFlt-1 gene transfer on laser-induced CNV. Mouse eyes were treated with SeV-F/HN-SIV-Empty, -hPEDF or -hsFlt-1 at 2.5 × 10⁷ TU/ml by the remove procedure. ELISA revealed that sufficient levels of hPEDF (790.3 ± 96.1 ng/g protein) and hsFlt-1 (47.6 ± 10.0 ng/g protein) were obtained 2 weeks after vector injection, and were sustained for at least 3 months postinjection (Fig. 3A and B). We performed laser photocoagulation 2 weeks after vector injection, and assessed the area of CNV 2 weeks after the laser treatment. CNV size was significantly reduced with SeV-F/HN-SIV vector-mediated retinal delivery of hPEDF or hsFlt-1, compared with CNV size in the eyes untreated or treated with SeV-F/HN-SIV-Empty (p < 0.01; Fig. 3C and D). Treatment with SeV-F/HN-SIV-hPEDF or hsFlt-1 also showed potent suppression of CNV when laser photocoagulation was performed 3 months after vector injection (p < 0.01; Fig. 3F and G), suggesting potential long-term inhibitory effects on CNV. Next, we compared CNV size within and outside of the vector-injected area. Treatment with SeV-F/HN-SIV-hPEDF or -hsFlt-1 was associated with significant CNV suppression in both areas (p < 0.05; Fig. 3E and H).

FIG. 3.

Suppression of laser-induced choroidal neovascularization (CNV) by subretinal injection of SeV-F/HN-SIV-hPEDF or SeV-F/HN-SIV-hsFlt-1. (A and B) ELISA to detect (A) human pigment epithelium-derived factor (hPEDF) and (B) human soluble Fms-like tyrosine kinase-1 (hsFlt-1) protein 2 weeks and 3 months after vector injection (n = 5 or 6). (C–H) Representative CNV lesions of choroidal flat mounts and quantitative analysis of the CNV area. Laser photocoagulation was performed (C–E) 2 weeks or (F–H) 3 months after each vector injection (n = 20–28). Eyes left untreated and those treated with SeV-F/HN-SIV-Empty were used as controls. The graphs show CNV size in the total retinal area (solid columns; D and G) and in vector-injected areas (open columns) and noninjected areas (shaded columns; E and H). *p < 0.05, **p < 0.01 versus untreated eyes. Scale bars, 100 μm.

Assessment of retinal toxicity with long-term overexpression of antiangiogenic factors

To determine any potential adverse effects of long-term overexpression of antiangiogenic factors on the retina, we next performed a histological examination of SeV-F/HN-SIV-treated retinas 6 months after vector injection by the remove procedure. Eyes that had undergone 6 months of sustained overexpression of hPEDF showed no significant changes in retinal structure; however, 6 months of sustained overexpression of hsFlt-1 resulted in a significant loss of photoreceptors in the vector-injected area (p < 0.05; Fig. 4A and B). These findings suggest that long-term blockade of VEGF signaling may be deleterious for maintaining retinal homeostasis in adult mice.

FIG. 4.

Assessment of potential retinal toxicity resulting from gene transfer of PEDF or sFlt-1 in normal adult mice. (A) Histological findings of the vector-injected area in the retina of untreated mice, and of mice treated with SeV-F/HN-SIV-Empty, SeV-F/HN-SIV-hPEDF, or SeV-F/HN-SIV-hsFlt-1, 6 months after vector injection. GCL, ganglion cell layer. Scale bar, 25 μm. (B) Quantitative analysis of cells in the outer nuclear layer (ONL). The number of nuclei in the ONL per 250 μm was counted at six points along the horizontal meridian of the eye (n = 6 or 7). Region A4–A6 corresponds to the nasal hemisphere of the eye. *p < 0.05, **p < 0.01 versus untreated eyes.

In the adult mouse retina, VEGF-A is secreted basally from the RPE, and fetal liver kinase-1 (Flk-1)/VEGFR-2 is expressed on the endothelium of the choriocapillaris facing the RPE layer, which is suggestive of a paracrine interaction between the two tissues (Saint-Geniez et al., 2006). To analyze the changes in choroidal vasculature observed with long-term overexpression of sFlt-1, we performed ICG angiography by in vivo scanning laser ophthalmoscopy. Retinas treated with SeV-F/HN-SIV-hPEDF or -Empty showed no significant alteration of ICG fluorescence, compared with that of untreated retinas. In contrast, fluorescein-filling defects of choroidal vessels were observed in those retinas treated with SeV-F/HN-SIV-hsFlt-1 in the area corresponding to the vector-injected site (Fig. 5). Ultrastructural analysis by transmission electron microscopy revealed that there were choriocapillaris vessels filled with densely packed, malformed erythrocytes with adjoining thrombocytes 1 month after SeV-F/HN-SIV-hsFlt-1 treatment (Fig. 6A and B). These findings were not observed in retinas untreated or treated with SeV-F/HN-SIV-Empty or -hPEDF (Fig. 6C and data not shown). No alteration of choriocapillaris endothelial cell fenestrations was observed between the groups (Fig. 6D). Ultrastructural signs of apoptosis of endothelial cells or RPE cells were not evident (data not shown). These findings indicate that long-term retinal delivery of PEDF is a safe and effective approach to suppressing CNV, but that long-term sFlt-1 expression may disturb the homeostatic balance of the retina, resulting in a disruption of the choroidal circulation and photoreceptor degeneration.

FIG. 5.

In vivo imaging of choroidal vessels by indocyanine green (ICG) angiography. Shown are ICG angiograms of the vector-injected area of mouse retinas 6 months after treatment with SeV-F/HN-SIV-Empty, SeV-F/HN-SIV-hPEDF, or SeV-F/HN-SIV-hsFlt-1 (n = 4 each). Eyes of untreated mice were used as a control. Arrows indicate defects in choroidal circulation in a retina treated with SeV-F/HN-SIV-hsFlt-1.

FIG. 6.

Ultrastructural analysis of the choriocapillaris by transmission electron microscopy. (A–C) Electron microscopy of the choriocapillaris in vector-injected areas 1 month after treatment with (A and B) SeV-F/HN-SIV-hsFlt-1 or (C) SeV-F/HN-SIV-Empty. Erythrocytes (E) and thrombocytes (Tr) were densely packed in the choriocapillaris lumen (A and B). BM, Bruch's membrane; En, endothelial cell. Scale bars, 5 μm. (D) Endothelial cell fenestrations of the choriocapillaris in retina untreated or treated with SeV-F/HN-SIV-Empty, SeV-F/HN-SIV-hPEDF, or SeV-F/HN-SIV-hsFlt-1. No alteration of choriocapillaris fenestrations was observed between the groups. Scale bars, 1 μm.

Discussion

In the present study, we characterized novel SIV-based lentiviral vectors pseudotyped with SeV-F and SeV-HN for retinal gene transfer. The key observations made in this study are as follows: (1) a brief vector–cell interaction period was sufficient for the SeV-F/HN-SIV vectors to achieve efficient gene transfer into RPE cells; (2) transgene expression mediated by SeV-F/HN-SIV vectors was stable and sustained over a 1-year period; (3) SeV-F/HN-SIV vector-mediated retinal gene transfer of hPEDF or hsFlt-1 substantially suppressed experimental CNV in mice; and (4) long-term overexpression of hPEDF did not exert any significant deleterious effects on the retinal tissue, whereas the long-term overexpression of hsFlt-1 resulted in photoreceptor degeneration in association with choroidal circulation defects. The rapid transduction ability of SeV-F/HN-SIV vectors is in clear contrast to reported findings obtained with conventional VSV-G-pseudotyped lentiviral vectors, rAAV, or adenoviral vectors (Teramoto et al., 1998; Masaki et al., 2001). To the best of our knowledge, this is the first report to demonstrate a limitation of the use of sFlt-1 for retinal gene therapy.

In this study, we demonstrated that the conventional VSV-G-pseudotyped SIV vectors required more than 24 hr of interaction time to achieve maximal gene transfer. rAAV and adenoviral vectors, which have been used in clinical trials of gene therapy for retinal diseases, also showed an interaction time-dependent increase in transgene expression and required more than 12 hr to reach the maximal expression level (Maeda et al., 1998; Teramoto et al., 1998). In contrast, the novel SIV vectors pseudotyped with SeV-F and SeV-HN achieved high-level gene transfer within several minutes, both in vitro and in vivo, as seen in previous reports using rSeV vectors (Ikeda et al., 2002). This unique feature of SeV-F/HN-SIV vectors enables the removal of subretinal vector solution and the resolution of RD during gene transfer surgery. Although subretinal injection of rAAV-RPE65 at the doses used in clinical studies has been shown to be safe in dogs and nonhuman primates (Jacobson et al., 2006a,b), retinal thinning was observed after subretinal injection of rAAV-RPE65 in one of three patients (Hauswirth et al., 2008). Of note, optical coherence tomography showed that the thickness of the outer nuclear layer was especially reduced in this patient. Retinal neurons, which are chronically stressed by degenerative retinopathy, may be more vulnerable to environmental changes. It is premature to assess the complications associated with subretinal viral vector injection, due to the small number of patients treated; however, we believe that the remove technique may reduce the nutrient starvation in outer retinal cells caused by RD and may provide safer retinal gene transfer. In addition, SeV-F/HN-SIV vectors exhibited stable and long-term transgene expression in the RPE over a 1-year period. In the clinical setting, most cases of AMD progress over several years, and long-lasting therapeutic effects are required for treatment. One limitation of the currently available anti-VEGF drugs is their short half-life in the eye and the need for repeated injections (Bakri et al., 2007). Therefore, SeV-F/HN-SIV vector-mediated continuous delivery of therapeutic proteins would be an attractive approach for the long-term inhibition of CNV in AMD patients.

Clinical studies have demonstrated the beneficial therapeutic effects of intravitreal injection of anti-VEGF-A drugs such as pegaptanib (an aptamer specific for VEGF-A₁₆₅) and ranibizumab (an Fab fragment of a humanized monoclonal pan-VEGF-A antibody) (Gragoudas et al., 2004; Brown et al., 2006; Rosenfeld et al., 2006). However, some investigators have raised concerns about the safety of long-term intraocular VEGF-A neutralization (Sang and D'Amore, 2008). In this study, we delivered the sFlt-1 gene into the RPE of the mouse retina via subretinal injection of SeV-F/HN-SIV-hsFlt-1, and we demonstrated that 6-month sustained overexpression of sFlt-1 resulted in photoreceptor degeneration. Although we previously demonstrated that subretinal injection of high-titer VSV-G-SIV vector (2.5 × 10⁸ TU/ml) induced sustained inflammation around the RPE and caused retinal degeneration (Ikeda et al., 2003), such inflammatory reaction and retinal damage were not observed in retinas treated with SeV-F/HN-SIV-Empty or -hPEDF at 2.5 × 10⁷ TU/ml, or in retinas treated with VSV-G-SIV vector at this dose (Ikeda et al., 2003), suggesting that overexpression of sFlt-1 is a major mediator of photoreceptor degeneration after SeV-F/HN-sFlt-1 treatment. In line with our data, Saint-Geniez and colleagues demonstrated that systemic VEGF neutralization by adenoviral vector-mediated overexpression of sFlt-1 induced the apoptosis of retinal neuronal cells, including photoreceptors (Saint-Geniez et al., 2008). However, there are several contradictory reports on the retinal effect of sFlt-1. Lai and colleagues and Pechan and colleagues demonstrated the long-term safety of rAAV vector-mediated retinal gene transfer of sFlt-1 (Lai et al., 2005; Pechan et al., 2009). One reason for these differences might involve differences in the levels of sFlt expression, because hsFlt-1 protein levels achieved with rAAV vectors were substantially lower than those observed in our study and in the study by Saint-Geniez and colleagues. In another study, Ueno and colleagues generated transgenic mice with doxycycline-inducible expression of sFlt-1 in their photoreceptors, and no significant changes in retinal structure or function were observed after a 7-month period of doxycycline treatment (Ueno et al., 2008). The reasons for these discrepant results remain unclear, but differences between cells expressing sFlt-1 may account for such discrepancies. Because VEGF-A is secreted basally from the RPE (Blaauwgeers et al., 1999), sFlt-1 expressed by the RPE may more effectively sequester VEGF-A and disrupt the homeostatic balance of the RPE–choriocapillary complex. These retinal effects of sFlt-1 are not necessarily identical to those of anti-VEGF-A drugs, because sFlt-1 binds not only to VEGF-A, but also to other VEGF family members such as VEGF-B, which exerts a potent neuroprotective effect on retinal neurons (Takahashi and Shibuya, 2005; Li et al., 2008). However, long-term follow-up of patients administered treatment with anti-VEGF-A drugs is required to monitor the eyes for retinal toxicity, as intravitreal injection of bevacizumab is known to cause mitochondrial swelling and disruption of the cristae in the photoreceptors of rabbit eyes (Inan et al., 2007).

The mechanisms of photoreceptor loss after VEGF neutralization remain unknown. One possible explanation would be that sFlt-1 may block the neuroprotective signaling of VEGF-A in photoreceptors. Nishijima and colleagues reported that VEGF-A directly protected retinal neurons in the ganglion cell layer and inner nuclear layer after ischemic reperfusion injury via the activation of Flk-1 (Nishijima et al., 2007). However, it remains unclear whether or not VEGF-A also provides direct neuroprotection to photoreceptors, because the levels of expression of VEGF receptors in photoreceptors were significantly lower than those in inner retinal neurons (Stitt et al., 1998; Nishijima et al., 2007; Li et al., 2008). As an alternative possibility, the impairment of choroidal circulation, which provides vascular support for photoreceptors and RPE cells, might induce photoreceptor degeneration. Flk-1 is strongly expressed in the endothelium of the choriocapillaris facing the RPE layer in the adult mouse retina (Saint-Geniez et al., 2006). In this study, we showed that persistent expression of sFlt-1 in the RPE resulted in a disruption of choroidal circulation, which suggests that VEGF signaling plays an essential role in maintaining the choroidal circulation. This explanation may also be supported by the finding that conditional inactivation of VEGF-A expression in the RPE layer resulted in an absence of choroidal vessels, disorganization of photoreceptors, and loss of visual function (Marneros et al., 2005). In ultrastructural analysis, we found choriocapillaris vessels filled with packed erythrocytes and thrombocytes after sFlt-1 gene transfer. These findings were similar to those made after intravitreal bevacizumab treatment in nonhuman primates (Peters et al., 2007), and suggest that these obstructions of the choriocapillaris might be a cause leading to choroidal circulation defects. No alteration of choriocapillaris endothelial cell fenestrations was observed in this study. Although Peters and colleagues reported early reduction of fenestrations after bevacizumab treatment, the reduction was recovered transiently (Peters et al., 2007). These changes in fenestrations were not observed after sustained systemic or local overexpression of sFlt-1 (Saint-Geniez et al., 2008; Ueno et al., 2008), suggesting that the loss of fenestrations might be a transient effect and would not be detectable at later time points.

Retinal gene transfer of PEDF by SeV-F/HN-SIV vectors efficiently inhibited experimental CNV at a level equivalent to that seen with SeV-F/HN-SIV-hsFlt-1, and did not exert any toxic effects on the normal retinal tissue. It has been demonstrated that PEDF induces the apoptosis of stimulated endothelial cells, but not that of quiescent endothelial cells, by targeting Fas/CD95 and the nuclear factor of activated T cells upregulated or activated by VEGF-A (Volpert et al., 2002; Zaichuk et al., 2004). Using the same mechanism, PEDF also induces the apoptosis of endothelial cells stimulated by other angiogenic factors such as basic fibroblast growth factor. Taken together, our findings and those of previous reports indicate that PEDF may specifically target activated endothelial cells during neovascularization, without affecting mature existing vessels. In addition to its antiangiogenic effects, PEDF has a neuroprotective effect on retinal neuronal cells (Miyazaki et al., 2003; Takita et al., 2003). We reported that PEDF directly inhibited photoreceptor apoptosis by regulating the mitochondrial release of apoptosis-inducing factor via Bcl-2 upregulation (Murakami et al., 2008a). These results suggest that gene therapy strategies using PEDF, which possesses both antiangiogenic and neuroprotective abilities, may be safe and effective for the treatment of retinal neovascular and degenerative diseases.

In conclusion, we have demonstrated that novel SIV vectors pseudotyped with SeV-F and SeV-HN showed rapid and efficient gene transfer to the RPE; thus, this system would enable the removal of subretinal vector solution shortly after vector injection. The long-term retinal delivery of PEDF by SeV-F/HN-SIV vectors was safe and effective at suppressing experimental CNV, whereas long-term delivery of sFlt-1 led to a disruption of the choroidal circulation and photoreceptor degeneration. These findings indicate that retinal gene therapy using PEDF may be a useful therapeutic strategy for the long-term management of CNV in AMD patients with a higher safety profile.

Footnotes

Acknowledgments

This work was supported in part by the Japanese Ministry of Education, Culture, Sports, Science, and Technology (grants-in-aid 20791259, 18390115, and 19791277 to Y.I., Y.Y., and M.M.). The authors are grateful to Drs. Eiji Akiba, Katsuyuki Mitomo, and Toshiaki Tabata for excellent technical assistance with vector construction and large-scale production. The authors also thank Mr. Hiroshi Fujii and Miss Chie Arimatsu for assistance with the experiments.

Author Disclosure Statement

Yoshikazu Yonemitsu is a member of the Scientific Advisory Board of the DNAVEC Corporation.

References

Acland

G.M.

, Aguirre

G.D.

, Ray

, Zhang

, Aleman

T.S.

, Cideciyan

A.V.

, Pearce-Kelling

S.E.

, Anand

, Zeng

, Maguire

A.M.

, Jacobson

S.G.

, Hauswirth

W.W.

, Bennett

2001. Gene therapy restores vision in a canine model of childhood blindness. Nat. Genet., 28:92–95.

Avery

R.L.

, Pieramici

D.J.

, Rabena

M.D.

, Castellarin

A.A.

, Nasir

M.A.

, Giust

M.J.

2006. Intravitreal bevacizumab (Avastin) for neovascular age-related macular degeneration. Ophthalmology, 113:363–372e365.

Bainbridge

J.W.

, Mistry

, De Alwis

, Paleolog

, Baker

, Thrasher

A.J.

, Ali

R.R.

2002. Inhibition of retinal neovascularisation by gene transfer of soluble VEGF receptor sFlt-1. Gene Ther., 9:320–326.

Bainbridge

J.W.

, Smith

A.J.

, Barker

S.S.

, Robbie

, Henderson

, Balaggan

, Viswanathan

, Holder

G.E.

, Stockman

, Tyler

, Petersen-Jones

, Bhattacharya

S.S.

, Thrasher

A.J.

, Fitzke

F.W.

, Carter

B.J.

, Rubin

G.S.

, Moore

A.T.

, Ali

R.R.

2008. Effect of gene therapy on visual function in Leber's congenital amaurosis. N. Engl. J. Med., 358:2231–2239.

Bakri

S.J.

, Snyder

M.R.

, Reid

J.M.

, Pulido

J.S.

, Ezzat

M.K.

, Singh

R.J.

2007. Pharmacokinetics of intravitreal ranibizumab (Lucentis) Ophthalmology, 114:2179–2182.

Blaauwgeers

H.G.

, Holtkamp

G.M.

, Rutten

, Witmer

A.N.

, Koolwijk

, Partanen

T.A.

, Alitalo

, Kroon

M.E.

, Kijlstra

, Van Hinsbergh

V.W.

, Schlingemann

R.O.

1999. Polarized vascular endothelial growth factor secretion by human retinal pigment epithelium and localization of vascular endothelial growth factor receptors on the inner choriocapillaris: Evidence for a trophic paracrine relation. Am. J. Pathol., 155:421–428.

Brown

D.M.

, Kaiser

P.K.

, Michels

, Soubrane

, Heier

J.S.

, Kim

R.Y.

, Sy

J.P.

, Schneider

2006. Ranibizumab versus verteporfin for neovascular age-related macular degeneration. N. Engl. J. Med., 355:1432–1444.

Campochiaro

P.A.

, Nguyen

Q.D.

, Shah

S.M.

, Klein

M.L.

, Holz

, Frank

R.N.

, Saperstein

D.A.

, Gupta

, Stout

J.T.

, Macko

, Dibartolomeo

, Wei

L.L.

2006. Adenoviral vector-delivered pigment epithelium-derived factor for neovascular age-related macular degeneration: Results of a phase I clinical trial. Hum. Gene Ther., 17:167–176.

Cideciyan

A.V.

, Hauswirth

W.W.

, Aleman

T.S.

, Kaushal

, Schwartz

S.B.

, Boye

S.L.

, Windsor

E.A.

, Conlon

T.J.

, Sumaroka

, Pang

J.J.

, Roman

A.J.

, Byrne

B.J.

, Jacobson

S.G.

2009. Human RPE65 gene therapy for Leber congenital amaurosis: Persistence of early visual improvements and safety at 1 year. Hum. Gene Ther., 20:999–1004.

10.

Darland

D.C.

, Massingham

L.J.

, Smith

S.R.

, Piek

, Saint-Geniez

, D'Amore

P.A.

2003. Pericyte production of cell-associated VEGF is differentiation-dependent and is associated with endothelial survival. Dev. Biol., 264:275–288.

11.

Dawson

D.W.

, Volpert

O.V.

, Gillis

, Crawford

S.E.

, Xu

, Benedict

, Bouck

N.P.

1999. Pigment epithelium-derived factor: A potent inhibitor of angiogenesis. Science, 285:245–248.

12.

Folkman

1995. Angiogenesis in cancer, vascular, rheumatoid and other disease. Nat. Med., 1:27–31.

13.

Friedman

D.S.

, O'Colmain

B.J.

, MunoZ

, Tomany

S.C.

, Mccarty

, De Jong

P.T.

, Nemesure

, Mitchell

, Kempen

2004. Prevalence of age-related macular degeneration in the United States. Arch. Ophthalmol., 122:564–572.

14.

Gehlbach

, Demetriades

A.M.

, Yamamoto

, Deering

, Xiao

W.H.

, Duh

E.J.

, Yang

H.S.

, Lai

, Kovesdi

, Carrion

, Wei

, Campochiaro

P.A.

2003. Periocular gene transfer of sFlt-1 suppresses ocular neovascularization and vascular endothelial growth factor-induced breakdown of the blood–retinal barrier. Hum. Gene Ther., 14:129–141.

15.

Gragoudas

E.S.

, Adamis

A.P.

, Cunningham

E.T.

Jr. , Feinsod

, Guyer

D.R.

2004. Pegaptanib for neovascular age-related macular degeneration. N. Engl. J. Med., 351:2805–2816.

16.

Hauswirth

W.W.

, Aleman

T.S.

, Kaushal

, Cideciyan

A.V.

, Schwartz

S.B.

, Wang

, Conlon

T.J.

, Boye

S.L.

, Flotte

T.R.

, Byrne

B.J.

, Jacobson

S.G.

2008. Treatment of Leber congenital amaurosis due to RPE65 mutations by ocular subretinal injection of adeno-associated virus gene vector: Short-term results of a phase I trial. Hum. Gene Ther., 19:979–990.

17.

Honda

, Sakamoto

, Ishibashi

, Inomata

, Ueno

2000. Experimental subretinal neovascularization is inhibited by adenovirus-mediated soluble VEGF/flt-1 receptor gene transfection: A role of VEGF and possible treatment for SRN in age-related macular degeneration. Gene Ther., 7:978–985.

18.

Hurwitz

, Saini

2006. Bevacizumab in the treatment of metastatic colorectal cancer: Safety profile and management of adverse events. Semin. Oncol., 33:S26–S34.

19.

Ikeda

, Yonemitsu

, Sakamoto

, Ishibashi

, Ueno

, Kato

, Nagai

, Fukumura

, Inomata

, Hasegawa

, Sueishi

2002. Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: Efficient gene transfer by brief exposure. Exp. Eye Res., 75:39–48.

20.

Ikeda

, Goto

, Yonemitsu

, Miyazaki

, Sakamoto

, Ishibashi

, Tabata

, Ueda

, Hasegawa

, Tobimatsu

, Sueishi

2003. Simian immunodeficiency virus-based lentivirus vector for retinal gene transfer: A preclinical safety study in adult rats. Gene Ther., 10:1161–1169.

21.

Inan

U.U.

, Avci

, Kusbeci

, Kaderli

, Avci

, Temel

S.G.

2007. Preclinical safety evaluation of intravitreal injection of full-length humanized vascular endothelial growth factor antibody in rabbit eyes. Invest. Ophthalmol. Vis. Sci., 48:1773–1781.

22.

Ishibashi

, Hata

, Yoshikawa

, Nakagawa

, Sueishi

, Inomata

1997. Expression of vascular endothelial growth factor in experimental choroidal neovascularization. Graefes Arch. Clin. Exp. Ophthalmol., 235:159–167.

23.

Jacobson

S.G.

, Acland

G.M.

, Aguirre

G.D.

, Aleman

T.S.

, Schwartz

S.B.

, Cideciyan

A.V.

, Zeiss

C.J.

, Komaromy

A.M.

, Kaushal

, Roman

A.J.

, Windsor

E.A.

, Sumaroka

, Pearce-Kelling

S.E.

, Conlon

T.J.

, Chiodo

V.A.

, Boye

S.L.

, Flotte

T.R.

, Maguire

A.M.

, Bennett

, Hauswirth

W.W.

2006a. Safety of recombinant adeno-associated virus type 2-RPE65 vector delivered by ocular subretinal injection. Mol. Ther., 13:1074–1084.

24.

Jacobson

S.G.

, Boye

S.L.

, Aleman

T.S.

, Conlon

T.J.

, Zeiss

C.J.

, Roman

A.J.

, Cideciyan

A.V.

, Schwartz

S.B.

, Komaromy

A.M.

, Doobrajh

, Cheung

A.Y.

, Sumaroka

, Pearce-Kelling

S.E.

, Aguirre

G.D.

, Kaushal

, Maguire

A.M.

, Flotte

T.R.

, Hauswirth

W.W.

2006b. Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis. Hum. Gene Ther., 17:845–858.

25.

Janssen

, Hoellenriegel

, Fogarasi

, Schrewe

, Seeliger

, Tamm

, Ohlmann

, May

C.A.

, Weber

B.H.

, Stohr

2008. Abnormal vessel formation in the choroid of mice lacking tissue inhibitor of metalloprotease-3. Invest. Ophthalmol. Vis. Sci., 49:2812–2822.

26.

Kendall

R.L.

, Wang

, Thomas

K.A.

1996. Identification of a natural soluble form of the vascular endothelial growth factor receptor, FLT-1, and its heterodimerization with KDR. Biochem. Biophys. Res. Commun., 226:324–328.

27.

Kobayashi

, Iida

, Ueda

, Hasegawa

2003. Pseudotyped lentivirus vectors derived from simian immunodeficiency virus SIVagm with envelope glycoproteins from paramyxovirus. J. Virol., 77:2607–2614.

28.

Krzystolik

M.G.

, Afshari

M.A.

, Adamis

A.P.

, Gaudreault

, Gragoudas

E.S.

, Michaud

N.A.

, Li

, Connolly

, O'Neill

C.A.

, Miller

J.W.

2002. Prevention of experimental choroidal neovascularization with intravitreal anti-vascular endothelial growth factor antibody fragment. Arch. Ophthalmol., 120:338–346.

29.

Lai

C.M.

, Shen

W.Y.

, Brankov

, Lai

Y.K.

, Barnett

N.L.

, Lee

S.Y.

, Yeo

I.Y.

, Mathur

, Ho

J.E.

, Pineda

, Barathi

, Ang

C.L.

, Constable

I.J.

, Rakoczy

E.P.

2005. Long-term evaluation of AAV-mediated sFlt-1 gene therapy for ocular neovascularization in mice and monkeys. Mol. Ther., 12:659–668.

30.

Lai

Y.K.

, Shen

W.Y.

, Brankov

, Lai

C.M.

, Constable

I.J.

, Rakoczy

P.E.

2002. Potential long-term inhibition of ocular neovascularisation by recombinant adeno-associated virus-mediated secretion gene therapy. Gene Ther., 9:804–813.

31.

Lamb

R.A.

, Kolakofsky

Fields

B.N.

, Knipe

D.M.

, Howley

P.M.

1996. Paramyxoviridae: The viruses and their replication. Fields Virology. Lippincott-Raven: Philadelphia, PA, 1177–1204.

32.

Lee

, Chen

T.T.

, Barber

C.L.

, Jordan

M.C.

, Murdock

, Desai

, Ferrara

, Nagy

, Roos

K.P.

, Iruela-Arispe

M.L.

2007. Autocrine VEGF signaling is required for vascular homeostasis. Cell, 130:691–703.

33.

Levine

R.J.

, Maynard

S.E.

, Qian

, Lim

K.H.

, England

L.J.

, Yu

K.F.

, Schisterman

E.F.

, Thadhani

, Sachs

B.P.

, Epstein

F.H.

, Sibai

B.M.

, Sukhatme

V.P.

, Karumanchi

S.A.

2004. Circulating angiogenic factors and the risk of preeclampsia. N. Engl. J. Med., 350:672–683.

34.

, Zhang

, Nagai

, Tang

, Zhang

, Scotney

, Lennartsson

, Zhu

, Qu

, Fang

, Hua

, Matsuo

, Fong

G.H.

, Ding

, Cao

, Becker

K.G.

, Nash

, Heldin

C.H.

, Li

2008. VEGF-B inhibits apoptosis via VEGFR-1-mediated suppression of the expression of BH3-only protein genes in mice and rats. J. Clin. Invest., 118:913–923.

35.

Lopez

P.F.

, Grossniklaus

H.E.

, Lambert

H.M.

, Aaberg

T.M.

, Capone

Jr. , Sternberg

Jr. , L'Hernault

1991. Pathologic features of surgically excised subretinal neovascular membranes in age-related macular degeneration. Am. J. Ophthalmol., 112:647–656.

36.

Maeda

, Ikeda

, Shimpo

, Ueno

, Ogasawara

, Urabe

, Kume

, Takizawa

, Saito

, Colosi

, Kurtzman

, Shimada

, Ozawa

1998. Efficient gene transfer into cardiac myocytes using adeno-associated virus (AAV) vectors. J. Mol. Cell. Cardiol., 30:1341–1348.

37.

Maguire

A.M.

, Simonelli

, Pierce

E.A.

, Pugh

E.N.

Jr. , Mingozzi

, Bennicelli

, Banfi

, Marshall

K.A.

, Testa

, Surace

E.M.

, Rossi

, Lyubarsky

, Arruda

V.R.

, Konkle

, Stone

, Sun

, Jacobs

, Dell'Osso

, Hertle

, Ma

J.X.

, Redmond

T.M.

, Zhu

, Hauck

, Zelenaia

, Shindler

K.S.

, Maguire

M.G.

, Wright

J.F.

, Volpe

N.J.

, Mcdonnell

J.W.

, Auricchio

, High

K.A.

, Bennett

2008. Safety and efficacy of gene transfer for Leber's congenital amaurosis. N. Engl. J. Med., 358:2240–2248.

38.

Maharaj

A.S.

, Walshe

T.E.

, Saint-Geniez

, Venkatesha

, Maldonado

A.E.

, Himes

N.C.

, Matharu

K.S.

, Karumanchi

S.A.

, D'Amore

P.A.

2008. VEGF and TGF-β are required for the maintenance of the choroid plexus and ependyma. J. Exp. Med., 205:491–501.

39.

Marneros

A.G.

, Fan

, Yokoyama

, Gerber

H.P.

, Ferrara

, Crouch

R.K.

, Olsen

B.R.

2005. Vascular endothelial growth factor expression in the retinal pigment epithelium is essential for choriocapillaris development and visual function. Am. J. Pathol., 167:1451–1459.

40.

Masaki

, Yonemitsu

, Komori

, Ueno

, Nakashima

, Nakagawa

, Fukumura

, Kato

, Hasan

M.K.

, Nagai

, Sugimachi

, Hasegawa

, Sueishi

2001. Recombinant Sendai virus-mediated gene transfer to vasculature: A new class of efficient gene transfer vector to the vascular system. FASEB J., 15:1294–1296.

41.

Maynard

S.E.

, Min

J.Y.

, Merchan

, Lim

K.H.

, Li

, Mondal

, Libermann

T.A.

, Morgan

J.P.

, Sellke

F.W.

, Stillman

I.E.

, Epstein

F.H.

, Sukhatme

V.P.

, Karumanchi

S.A.

2003. Excess placental soluble Fms-like tyrosine kinase 1 (sFlt1) may contribute to endothelial dysfunction, hypertension, and proteinuria in preeclampsia. J. Clin. Invest., 111:649–658.

42.

Miyazaki

, Ikeda

, Yonemitsu

, Goto

, Sakamoto

, Tabata

, Ueda

, Hasegawa

, Tobimatsu

, Ishibashi

, Sueishi

2003. Simian lentiviral vector-mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats. Gene Ther., 10:1503–1511.

43.

Miyazaki

, Kiyohara

, Yoshida

, Iida

, Nose

, Ishibashi

2005. The 5-year incidence and risk factors for age-related maculopathy in a general Japanese population: The Hisayama Study. Invest. Ophthalmol. Vis. Sci., 46:1907–1910.

44.

Miyoshi

, Takahashi

, Gage

F.H.

, Verma

I.M.

1997. Stable and efficient gene transfer into the retina using an HIV-based lentiviral vector. Proc. Natl. Acad. Sci. U.S.A., 94:10319–10323.

45.

Mori

, Gehlbach

, Ando

, Mcvey

, Wei

, Campochiaro

P.A.

2002. Regression of ocular neovascularization in response to increased expression of pigment epithelium-derived factor. Invest. Ophthalmol. Vis. Sci., 43:2428–2434.

46.

Murakami

, Ikeda

, Yonemitsu

, Onimaru

, Nakagawa

, Kohno

, Miyazaki

, Hisatomi

, Nakamura

, Yabe

, Hasegawa

, Ishibashi

, Sueishi

2008a. Inhibition of nuclear translocation of apoptosis-inducing factor is an essential mechanism of the neuroprotective activity of pigment epithelium-derived factor in a rat model of retinal degeneration. Am. J. Pathol., 173:1326–1338.

47.

Murakami

, Ikeda

, Yonemitsu

, Tanaka

, Kondo

, Okano

, Kohno

, Miyazaki

, Inoue

, Hasegawa

, Ishibashi

, Sueishi

2008b. Newly-developed Sendai virus vector for retinal gene transfer: Reduction of innate immune response via deletion of all envelope-related genes. J. Gene Med., 10:165–176.

48.

Nishijima

, Ng

Y.S.

, Zhong

, Bradley

, Schubert

, Jo

, Akita

, Samuelsson

S.J.

, Robinson

G.S.

, Adamis

A.P.

, Shima

D.T.

2007. Vascular endothelial growth factor-A is a survival factor for retinal neurons and a critical neuroprotectant during the adaptive response to ischemic injury. Am. J. Pathol., 171:53–67.

49.

Pechan

, Rubin

, Lukason

, Ardinger

, Dufresne

, Hauswirth

W.W.

, Wadsworth

S.C.

, Scaria

2009. Novel anti-VEGF chimeric molecules delivered by AAV vectors for inhibition of retinal neovascularization. Gene Ther., 16:10–16.

50.

Peters

, Heiduschka

, Julien

, Ziemssen

, Fietz

, Bartz-Schmidt

K.U.

, Schraermeyer

2007. Ultrastructural findings in the primate eye after intravitreal injection of bevacizumab. Am. J. Ophthalmol., 143:995–1002.

51.

Rosenfeld

P.J.

, Brown

D.M.

, Heier

J.S.

, Boyer

D.S.

, Kaiser

P.K.

, Chung

C.Y.

, Kim

R.Y.

2006. Ranibizumab for neovascular age-related macular degeneration. N. Engl. J. Med., 355:1419–1431.

52.

Saint-Geniez

, Maldonado

A.E.

, D'Amore

P.A.

2006. VEGF expression and receptor activation in the choroid during development and in the adult. Invest. Ophthalmol. Vis. Sci., 47:3135–3142.

53.

Saint-Geniez

, Maharaj

A.S.

, Walshe

T.E.

, Tucker

B.A.

, Sekiyama

, Kurihara

, Darland

D.C.

, Young

M.J.

, D'Amore

P.A.

2008. Endogenous VEGF is required for visual function: Evidence for a survival role on muller cells and photoreceptors. PLoS ONE, 3:e3554.

54.

Sang

D.N.

, D'Amore

P.A.

2008. Is blockade of vascular endothelial growth factor beneficial for all types of diabetic retinopathy? Diabetologia, 51:1570–1573.

55.

Steele

F.R.

, Chader

G.J.

, Johnson

L.V.

, Tombran-Tink

1993. Pigment epithelium-derived factor: Neurotrophic activity and identification as a member of the serine protease inhibitor gene family. Proc. Natl. Acad. Sci. U.S.A., 90:1526–1530.

56.

Stitt

A.W.

, Simpson

D.A.

, Boocock

, Gardiner

T.A.

, Murphy

G.M.

, Archer

D.B.

1998. Expression of vascular endothelial growth factor (VEGF) and its receptors is regulated in eyes with intra-ocular tumours. J. Pathol., 186:306–312.

57.

Takahashi

, Shibuya

2005. The vascular endothelial growth factor (VEGF)/VEGF receptor system and its role under physiological and pathological conditions. Clin. Sci., 109:227–241.

58.

Takita

, Yoneya

, Gehlbach

P.L.

, Duh

E.J.

, Wei

L.L.

, Mori

2003. Retinal neuroprotection against ischemic injury mediated by intraocular gene transfer of pigment epithelium-derived factor. Invest. Ophthalmol. Vis. Sci., 44:4497–4504.

59.

Taniwaki

, Becerra

S.P.

, Chader

G.J.

, Schwartz

J.P.

1995. Pigment epithelium-derived factor is a survival factor for cerebellar granule cells in culture. J. Neurochem., 64:2509–2517.

60.

Teramoto

, Bartlett

J.S.

, Mccarty

, Xiao

, Samulski

R.J.

, Boucher

R.C.

1998. Factors influencing adeno-associated virus-mediated gene transfer to human cystic fibrosis airway epithelial cells: Comparison with adenovirus vectors. J. Virol., 72:8904–8912.

61.

Tombran-Tink

, Johnson

L.V.

1989. Neuronal differentiation of retinoblastoma cells induced by medium conditioned by human RPE cells. Invest. Ophthalmol. Vis. Sci., 30:1700–1707.

62.

Tsutsumi

, Sonoda

K.H.

, Egashira

, Qiao

, Hisatomi

, Nakao

, Ishibashi

, Charo

I.F.

, Sakamoto

, Murata

, Ishibashi

2003. The critical role of ocular-infiltrating macrophages in the development of choroidal neovascularization. J. Leukoc. Biol., 74:25–32.

63.

Ueno

, Pease

M.E.

, Wersinger

D.M.

, Masuda

, Vinores

S.A.

, Licht

, Zack

D.J.

, Quigley

, Keshet

, Campochiaro

P.A.

2008. Prolonged blockade of VEGF family members does not cause identifiable damage to retinal neurons or vessels. J. Cell Physiol., 217:13–22.

64.

Volpert

O.V.

, Zaichuk

, Zhou

, Reiher

, Ferguson

T.A.

, Stuart

P.M.

, Amin

, Bouck

N.P.

2002. Inducer-stimulated Fas targets activated endothelium for destruction by anti-angiogenic thrombospondin-1 and pigment epithelium-derived factor. Nat. Med., 8:349–357.

65.

Yonemitsu

, Kitson

, Ferrari

, Farley

, Griesenbach

, Judd

, Steel

, Scheid

, Zhu

, Jeffery

P.K.

, Kato

, Hasan

M.K.

, Nagai

, Masaki

, Fukumura

, Hasegawa

, Geddes

D.M.

, Alton

E.W.

2000. Efficient gene transfer to airway epithelium using recombinant Sendai virus. Nat. Biotechnol., 18:970–973.

66.

Zaichuk

T.A.

, Shroff

E.H.

, Emmanuel

, Filleur

, Nelius

, Volpert

O.V.

2004. Nuclear factor of activated T cells balances angiogenesis activation and inhibition. J. Exp. Med., 199:1513–1522.