Activation of the extracellular signal-regulated kinase 1/2 pathway by AAV gene transfer protects retinal ganglion cells in glaucoma.

Glaucoma is the second leading cause of blindness in the world. Loss of vision in glaucomatous optic neuropathy is caused by the selective degeneration of retinal ganglion cells (RGCs). Ocular hypertension is a major risk factor in glaucoma, but visual field defects continue to progress in some patients despite the use of drugs that lower intraocular pressure. At present, there are no effective neuroprotective strategies for the treatment of this disease. The extracellular signal-regulated kinase (Erk) 1/2 pathway is an evolutionarily conserved mechanism used by several peptide factors to promote cell survival. Here we tested if selective activation of Erk1/2 protected RGCs in a rat model of experimental glaucoma. We used recombinant adeno-associated virus to transduce RGCs with genes encoding constitutively active or wild-type MEK1 (approved gene symbol MAP2K1), the upstream activator of Erk1/2. MEK1 gene transfer into RGCs markedly increased neuronal survival: 1366 +/- 70 RGCs/mm(2) (mean +/- SEM) were alive in the dorsal retina at 5 weeks after ocular hypertension surgery, a time when only 680 +/- 86 RGCs/mm(2) of these neurons remained in control eyes. We conclude that the Erk1/2 pathway plays a key role in the protection of RGCs from ocular hypertensive damage. This study identifies a novel gene therapy strategy in which selective activation of the Erk1/2 signaling pathway effectively slows cell death in glaucoma.     

A vitrectomy improves the transfection efficiency of adenoviral vector-mediated gene transfer to Müller cells.

The neural retina is a logical target of gene therapy for various ocular diseases. We developed a new gene delivery method to the neural retina using an adenoviral vector with a high degree of gene transfection efficiency and less functional damage. An adenoviral vector bearing the lacZ gene (AdCALacZ) was injected into the eyes of adult Wistar rats after an SF6 compression gas vitrectomy and left for 30 min followed by washing with balanced salt solution (BSS) (method A). Three other methods, comprising a simple intravitreal injection (method B), an intravitreal injection after an SF6 compression gas vitrectomy (method C) or a subretinal injection (method D), were also studied. The gene expression was examined 6 days after the AdCALacZ injection. An immunohistochemical study for antivimentin, antiglial fibrillary acidic protein and anti S100 protein antibodies showed the neural retinal cells (Müller cells) to be primarily transfected by methods A, B and C, while only a few cells were transfected by method D. The expression of beta-galactosidase was visualized by X-gal staining and the positive areas on each hemiflat mount specimen were measured by an image analyzer and then were adopted as a value of gene transfer efficiency. The highest degree of gene expression was obtained by methods A (23.2% of total retinal area) and C (19.8%), while the lowest degree was obtained by method B (8.9%). The inflammation was observed in all eyes and the value of inflammation was quantified as the average inflammatory cell number in each microscopic field (cells per fields). A moderate degree of inflammation was induced by methods B (28.3 cells per field) and C (27.5 cells per field) and a minimal degree of inflammation was induced by method A (11.2 cells per field). We evaluated the retinal function by measuring an electroretinogram (ERG). The amplitudes of the ERG were depressed in all eyes treated with AdCALacZ. This depression was manifested most by methods B and C, and least by method A. The deterioration in the ERG findings seemed to correlate with the intensity of inflammation. Our study showed that an intravitreal injection with an adenoviral vector can transfer the genes to the neural retinal cells and therefore a vitrectomy and the subsequent removal of the adenoviral vector, can thus significantly improve the transfection efficiency and also reduce the degree of functional damage.     

Baculoviral IAP Repeat-Containing-4 Protects Optic Nerve Axons in a Rat Glaucoma Model

Gene therapy represents an attractive approach for the treatment of eye diseases such as glaucoma. Ocular administration of viral vectors produces localized retinal gene expression with reduced risks of side effects reported with systemic administration of viral vectors. Recombinant adeno-associated viral (AAV) vectors have proven effective in producing long-term retinal gene expression, due to stable integration of DNA into the genome and lack of host immune response to the virus. Recently developed AAV constructs using the chicken β-actin (CBA) promoter drive highly efficient transgene expression in retinal ganglion cells (RGCs), photoreceptors, and pigment epithelium. Rats were given unilateral intravitreal injections of AAV-CBA vector coding for human baculoviral IAP repeat-containing protein-4 (BIRC4), a potent caspase inhibitor. Ocular hypertension was induced in the same eye by sclerosis of aqueous humor outflow channels. After chronic exposure to elevated intraocular pressure, we performed optic nerve axon counts to determine the neuroprotective effects of retinal BIRC4 expression, and compared axon survival with vector and balanced salt solution control groups. Gene therapy delivering BIRC4 significantly promoted optic nerve axon survival in a chronic ocular hypertensive model of rat glaucoma. Blocking RGC apoptosis with caspase inhibitors represents a promising approach for treatment of human glaucoma.     

Simian lentiviral vector-mediated retinal gene transfer of pigment epithelium-derived factor protects retinal degeneration and electrical defect in Royal College of Surgeons rats

Retinitis pigmentosa (RP) is a heterogenous group of inherited retinal diseases resulting in adult blindness caused by mutations of various genes. Although it is difficult to cure the blindness that results from these diseases, delaying the disease progression may be of great benefit, since the majority of RP diseases are seen in middle age or later. To test a gene therapy strategy for RP using a neurotrophic factor gene, we assessed the effect of simian lentivirus (SIV)-mediated subretinal gene transfer of pigment epithelium-derived factor (PEDF), a potent neurotrophic factor, during the disease progression in Royal College of Surgeons (RCS) rats, a well-accepted animal model of RP. Regional gene transfer via SIV into the peripheral subretinal space at the nasal hemisphere was performed in all animals to monitor site-specific transgene expression as well as the therapeutic effect in each retina. Gene transfer of lacZ and PEDF was observed in the regional pigment epithelium corresponding to the regional gene transfer. Histologically, PEDF gene transfer significantly protected the loss of photoreceptor cells (PCs) corresponding to the regions of the gene transfer, compared to those of control groups during the course of the experiment. The antiapoptotic effect of PEDF on PCs is likely to be a related mechanism, because a significant reduction of terminal dUTP-nicked end labeling-positive PC numbers was found in PEDF-treated eyes compared to those of the control group (P<0.05). PEDF-treated eyes also retained a significant sensitivity to light flash during the experimental course. These findings clearly show that neuroprotective gene therapy using PEDF can protect retinal degeneration and functional defects in individuals with RP.     

Immune responses to adeno-associated virus type 2 encoding channelrhodopsin-2 in a genetically blind rat model for gene therapy

We had previously reported that transduction of the channelrhodopsin-2 (ChR2) gene into retinal ganglion cells restores visual function in genetically blind, dystrophic Royal College of Surgeons (RCS) rats. In this study, we attempted to reveal the safety and influence of exogenous ChR2 gene expression. Adeno-associated virus (AAV) type 2 encoding ChR2 fused to Venus (rAAV-ChR2V) was administered by intra-vitreous injection to dystrophic RCS rats. However, rAAV-ChR2 gene expression was detected in non-target organs (intestine, lung and heart) in some cases. ChR2 function, monitored by recording visually evoked potentials, was stable across the observation period (64 weeks). No change in retinal histology and no inflammatory marker of leucocyte adhesion in the retinal vasculature were observed. Although antibodies to rAAV (0.01-12.21?μg?ml(-1)) and ChR2 (0-4.77?μg?ml(-1)) were detected, their levels were too low for rejection. T-lymphocyte analysis revealed recognition by T cells and a transient inflammation-like immune reaction only until 1 month after the rAAV-ChR2V injection. In conclusion, ChR2, which originates from Chlamydomonas reinhardtii, can be expressed without immunologically harmful reactions in vivo. These findings will help studies of ChR2 gene transfer to restore vision in progressed retinitis pigmentosa.     

Simian immunodeficiency virus-based lentivirus vector for retinal gene transfer: a preclinical safety study in adult rats

Although lentivirus vectors hold promise for ocular gene therapy, they also have potential safety issues, particularly in the case of the current human immunodeficiency virus-based vectors. We recently developed a novel lentivirus vector derived from the nonpathogenic simian immunodeficiency virus from African green monkeys (SIVagm) to minimize these potentials. In this preclinical study, we evaluated whether SIV vector could be efficiently and safely applicable to retinal gene transfer by assessing the transgene expression, retinal function and histology over a 1-year period following subretinal injection in adult rats. The functional assessment via electroretinogram after both titers of SIV-lacZ (2.5 x 10(7) or 2.5 x 10(8) transducing units/ml) injection revealed both the dark and light adaptations to soon be impaired, in a dose-dependent manner, after a buffer injection as well, and all of them recovered to the control range by day 30. In both titers tested, the retinas demonstrated a frequent transgene expression mainly in the retinal pigment epithelium; however, the other retinal cells rarely expressed the transgene. Retinas exposed to a low titer virus showed no significant inflammatory reaction throughout the observation period, and also maintained the transgene expression over a 1-year period. In the retinas exposed to a high titer virus, however, mononuclear cell infiltration persisted in the subretinal area, and the retina that corresponded to the injected area finally underwent degeneration by around day 90. No retinal neoplastic lesions could be found in any animals over the 1-year period. We thus propose that SIV-mediated stable gene transfer might be useful for ocular gene transfer; however, more attention should be paid to avoiding complications when administering high titer lentivirus to the retina.     

XIAP-mediated neuroprotection in retinal ischemia

Retinal ischemia results in the loss of vision in a number of ocular diseases including acute glaucoma, diabetic retinopathy, hypertensive retinopathy and retinal vascular occlusion. Recent studies have shown that most of the neuronal death that leads to loss of vision results from apoptosis. XIAP-mediated gene therapy has been shown to protect a number of neuronal types from apoptosis but has never been assessed in retinal neurons following ischemic-induced cell death. We injected an adeno-associated viral vector expressing XIAP or GFP into rat eyes and 6 weeks later, rendered them ischemic by raising intraocular pressure. Functional analysis revealed that XIAP-treated eyes retained larger b-wave amplitudes than GFP-treated eyes up to 4 weeks post-ischemia. The number of cells in the inner nuclear layer (INL) and the thickness of the inner retina were significantly preserved in XIAP-treated eyes compared to GFP-treated eyes. Similarly, there was no significant reduction in optic nerve axon numbers in XIAP-treated eyes. There were also significantly fewer TUNEL (TdT-dUTP terminal nick end labeling) positive cells in the INL of XIAP-treated retinas at 24 h post-ischemia. Thus, XIAP-mediated gene therapy imparts both functional and structural protection to the retina after a transient ischemic episode.     

Long-term rescue of retinal structure and function by rhodopsin RNA replacement with a single adeno-associated viral vector in P23H RHO transgenic mice

Many mutations in the human rhodopsin gene (RHO) cause autosomal dominant retinitis pigmentosa (ADRP). Our previous studies with a P23H (proline-23 substituted by histidine) RHO transgenic mouse model of ADRP demonstrated significant improvement of retinal function and preservation of retinal structure after transfer of wild-type rhodopsin by AAV. In this study we demonstrate long-term rescue of retinal structure and function by a single virus expressing both RHO replacement cDNA and small interfering RNA (siRNA) to digest mouse Rho and human P23H RHO mRNA. This combination should prevent overexpression of rhodopsin, which can be deleterious to photoreceptors. On the basis of the electroretinogram (ERG) response, degeneration of retinal function was arrested at 2 months postinjection, and the response was maintained at this level until termination at 9 months. Preservation of the ERG response in P23H RHO mice reflected survival of photoreceptors: both the outer nuclear layer (ONL) and outer segments of photoreceptor cells maintained the same thickness as in nontransgenic mice, whereas the control injected P23H eyes exhibited severe thinning of the ONL and outer segments. These findings suggest that delivery of both a modified cDNA and an siRNA by a single adeno-associated viral vector provided long-term rescue of ADRP in this model. Because the siRNA targets human as well as mouse rhodopsin mRNAs, the combination vector may be useful for the treatment of human disease.     

Sustained transduction of ocular cells with a bovine immunodeficiency viral vector

Human immunodeficiency viral (HIV) vectors mediate long-term transduction of many types of nondividing cells in vivo. Bovine immunodeficiency virus (BIV) is a lentivirus that shares many characteristics with HIV, but does not cause human disease. In this study, we investigated the potential of BIV vectors for ocular gene therapy. An enhanced green fluorescent protein (eGFP)-encoding reporter gene was packaged in recombinant BIV vector (BIV.eGFP). Adult C57BL/6 mice were given an intravitreous (5 x 10(4) or 5 x 10(5) transducing units [TU]) or subretinal (5 x 10(5) TU) injection of BIV.eGFP and then GFP expression was assessed at several time points. In vivo examinations of mice showed that subretinal injection of BIV.eGFP resulted in strong expression of GFP from the first examination at 1 week through the final examination at 20 weeks. Only a few mice that received intravitreous injection of BIV.eGFP showed GFP expression by ocular examinations until 11-12 weeks, when most showed small areas of expression. Postmortem examinations showed prominent GFP expression in retinal pigmented epithelial (RPE) cells throughout the region of subretinal injection of vector, although occasional negatively staining RPE cells were scattered among the much more numerous, brilliantly staining cells. Ciliary epithelial cells frequently expressed GFP, as did occasional Müller cells and rarely other retinal cells. The expression was stable from the first time point (2 weeks) to the last (20 weeks). Postmortem examination of eyes given an intravitreous injection of BIV.eGFP showed transduction of cells in the corneal endothelium and a few scattered retinal cells. There was no evidence of inflammation or toxicity in any eyes. These data show that BIV vectors mediate rapid and sustained transduction of RPE cells, suggesting that they may be useful for ocular gene therapy targeting RPE cells.     

Systemic adeno-associated virus-mediated gene therapy preserves retinal ganglion cells and visual function in DBA/2J glaucomatous mice

A slow progressive death of neurons is the hallmark of neurodegenerative diseases, such as glaucoma. A therapeutic candidate, erythropoietin (EPO), has shown promise in many models of these diseases; however, it also causes polycythemia, a potentially lethal side effect. We have developed a novel mutant form of EPO that is neuroprotective but no longer erythropoietic by altering a single amino acid (arginine to glutamate at position 76; R76E). We hypothesized that a single intramuscular injection of recombinant adeno-associated virus carrying EpoR76E (rAAV2/5.CMV.EpoR76E) would protect retinal ganglion cells in a mouse model of glaucoma without inducing polycythemia. This systemic treatment not only protected the retinal ganglion cell somata located within the retina; it also preserved axonal projections within the optic nerve, while maintaining the hematocrit within normal limits. The rescued retinal ganglion cells retained their visual function demonstrated by flash visual evoked potentials. To our knowledge, this is the first demonstration of a therapy that protects neurons from death and prevents loss of visual function from the slow neurodegenerative effects of glaucoma. Because of its broad range of cellular targets, EpoR76E is likely to be successful in treating other neurodegenerative diseases as well.     

AAV-mediated intravitreal gene therapy reduces lysosomal storage in the retinal pigmented epithelium and improves retinal function in adult MPS VII mice.

The beta-glucuronidase-deficient mucopolysaccharidosis type VII (MPS VII) mouse accumulates partially degraded glycosaminoglycans in many cell types, including retinal pigmented epithelial (RPE) cells in the eye. This lysosomal storage in RPE cells leads to progressive retinal degeneration and reduced function as measured by flash electroretinography (ERG). The impact of AAV-mediated intraocular gene therapy on pathology and retinal function was examined in normal and MPS VII mice treated at 4 weeks of age, when lysosomal storage is evident but functional impairment is minimal in affected animals. At 16 weeks, an age at which untreated MPS VII mice have advanced histologic lesions and significantly reduced ERG amplitudes, treated eyes had nearly normal levels of beta-glucuronidase activity, preservation of cells in the outer nuclear layer of the retina, and decreased lysosomal storage within the RPE. The AAV-treated MPS VII mice also had significantly increased dark-adapted ERG amplitudes compared to untreated MPS VII mice. Although retinal function was improved, the efficacy of the treatment depended heavily on parameters related to the injection procedure, such as the injection volume, injection site, and vector dose. These data suggest that intraocular AAV-mediated therapy may be efficacious for treating the retinal disease associated with certain lysosomal storage diseases.     

慢性高眼压模型大鼠血视网膜屏障损伤观察

目的观察慢性高眼压大鼠血视网膜屏障损伤情况。方法建立慢性高眼压大鼠模型,并检测眼压,分为A、B 2组。分别于1、4、8周时行股静脉注射30 g/L伊文思蓝,2 h后以温生理盐水灌注动物,立即摘除双侧眼球,A组行冰冻切片,荧光显微镜下观察;B组分离视网膜,称湿重,提取其内伊文思蓝,测光密度值,计算大鼠视网膜伊文思蓝渗透率。结果激光造模后大鼠眼压持续缓慢升高,与对照组存在显著差异;4周时眼压达最高值,为(24.8±3.2)mmHg;12周时眼压仍未见明显下降。激光后1、4、8周时荧光显微镜下观察视网膜内未见明显伊文思蓝渗透;1、4、8周时大鼠视网膜标准化EB含量分别为16.63±4.48、17.25±3.71、16.94±3.59(ng/mg),与对照组(12.16±3.32)ng/mg比较无显著差异。相关分析慢性高眼压大鼠视网膜通透率与眼压呈正的直线相关。结论慢性高眼压情况下尚不能破坏大鼠血视网膜屏障。     

准分子激光原位角膜磨镶术对视网膜功能的影响

目的 观察准分子激光原位角膜磨镶术（LASIK）对视网膜功能和视神经纤维层厚度的影响。方法 近视眼患者15例30眼，术前经严格检查后行LASIK。术前、术后1d、7d、1个月、3个月、6个月检查裸眼和矫正视力，散瞳验光测量屈光度，非接触眼压计测量眼压，AB超测量角膜厚度及眼轴长度．角膜地形图扫描，间接眼底镜散瞳下检查眼底，并行视网膜电流图、视觉诱发电位测定及光学相干断层扫描。结果 LASIK术后早期眼压无明显变化，视觉电生理无明显变化；术后1周，平均视神经纤维层厚度降低（P〈0．05），术后1～6个月神经纤维层厚度逐渐恢复至术前水平。结论 LASIK对近视眼患者术后的视网膜功能和视神经纤维层厚度无明显影响。     

Ocular cell transfection with the human basic fibroblast growth factor gene delays photoreceptor cell degeneration in RCS rats

Based on the K8/JTS-1-mediated transfection technique, we developed an in vivo protocol for an efficient transfer of plasmid DNA to ocular cells. As determined with condensed plasmids containing reporter genes for either beta-galactosidase (pcDNA-lacZ) or enhanced green fluorescent protein (pREP-EGFP), the immortalized human retinal epithelial cells RPE D407 and human embryonic kidney 293 cells can be transfected with typical efficiencies of 11 and 19%, respectively. Unlike 293 cells, RPE D407 cells had a reduced viability on transfection with both plasmids. In vivo, subretinal injections of DNA-K8/JTS-1 complexes revealed reporter gene expression in choroidal and RPE cells of normal pink-eyed Royal College of Surgeons (RCS) rats. The validity of this transfection technique in terms of retinal cell survival in RCS rats was then examined by using pREP-hFGF2 plasmid, which encodes the human basic fibroblast growth factor isoforms (hFGF2). Subretinal injection of pREP-hFGF2-K8/JTS-1 complexes into 3-week-old dystrophic RCS rat eyes reveals a delayed photoreceptor cell degeneration 60 days postinjection. In this case, the average analyzed field points with delayed cell dystrophy represent 14 to 17% of the retinal surface as compared with 2.6 and 4% in pREP5beta and vehicle-injected eyes, respectively. Peptide-mediated in oculo transfection thus appears to be a promising technique for the treatment of retinal cell and photoreceptor degenerations.     

Safety in nonhuman primates of ocular AAV2-RPE65, a candidate treatment for blindness in Leber congenital amaurosis

Leber congenital amaurosis (LCA) is a molecularly heterogeneous disease group that leads to blindness. LCA caused by RPE65 mutations has been studied in animal models and vision has been restored by subretinal delivery of AAV-RPE65 vector. Human ocular gene transfer trials are being considered. Our safety studies of subretinal AAV-2/2.RPE65 in RPE65-mutant dogs showed evidence of modest photoreceptor loss in the injection region in some animals at higher vector doses. We now test the hypothesis that there can be vectorrelated toxicity to the normal monkey, with its human-like retina. Good Laboratory Practice safety studies following single intraocular injections of AAV-2/2.RPE65 in normal cynomolgus monkeys were performed for 1-week and 3-month durations. Systemic toxicity was not identified. Ocular-specific studies included clinical examinations, electroretinography, and retinal histopathology. Signs of ocular inflammation postinjection had almost disappeared by 1 week. At 3 months, electroretinography in vector-injected eyes was no different than in vehicle-injected control eyes or compared with presurgical recordings. Healed sites of retinal perforation from subretinal injections were noted clinically and by histopathology. Foveal architecture in subretinally injected eyes, vector or vehicle, could be abnormal. Morphometry of central retina showed no photoreceptor layer thickness abnormalities occurring in a dose-dependent manner. Vector sequences were present in the injected retina, vitreous, and optic nerve at 1 week but not consistently in the brain. At 3 months, there were no vector sequences in optic nerve and brain. The results allow for consideration of an upper range for no observed adverse effect level in future human trials of subretinal AAV-2/2.RPE65. The potential value of foveal treatment for LCA and other retinal degenerations warrants further research into how to achieve gene transfer without retinal injury from surgical detachment of the retina.     

POD Nanoparticles Expressing GDNF Provide Structural and Functional Rescue of Light-induced Retinal Degeneration in an Adult Mouse

Peptide for ocular delivery (POD) is a novel cationic cell-penetrating peptide (CPP) which, when conjugated with polyethylene glycol (PEG-POD), can deliver plasmid DNA to the retinal pigment epithelium (RPE) of adult murine retina. PEG-POD nanoparticles containing an expression cassette for glial cell line–derived neurotrophic factor (PEG–POD~GDNF) were investigated for their ability to inhibit light-induced photoreceptor apoptosis. PEG-POD~GDNF, control nanoparticles, or buffer were injected into the subretinal space of adult murine retina and retinal degeneration induced by blue light. Animals injected with PEG-POD~GDNF showed a significant reduction (3.9–7.7 fold) in apoptosis relative to control-injected animals. The thickness of the outer nuclear layer (ONL) of the superior retina of PEG-POD~GDNF-injected eyes was significantly greater (23.6–39.3%) than control-injected retina 14 days post-light treatment. PEG-POD~GDNF-injected eyes showed a 27–39% greater functional response relative to controls, as measured by electroretinogram (ERG) 7 days post-light treatment. This is one of only two studies demonstrating histological and functional rescue of a mouse model of retinal degeneration following nonviral administration of a transgene into adult retina. Although rescue is short lived for clinical application, this study represents an important step in the development of nonviral gene therapy for retinal diseases.     

Monitoring the effect of mild ischemia with a built-in light-emitting diode contact lens electrode and a low-cost custom-made apparatus

Electroretinography (ERG) is widely used in clinical work and research to assess the retinal function. We evaluated an easy to build ERG setup adapted for small animals comprising two contact lens electrodes with a built-in light-emitting diode and a custom-made amplification system. The system's sensitivity was tested by monitoring ERG in albino rat eyes subjected to mild ischemia. Flash ERG was recorded by two contact lens electrodes positioned on the rat's corneas and used alternately as test or reference. The a- and b-wave amplitudes, a-wave latency, b-wave implicit time and oscillatory potentials (OPs) were analyzed. Ischemia was achieved by elevating the intraocular pressure in the eye's anterior chamber. ERG was recorded on post-ischemia (PI) days -1, 1, 3 and 7. Morphological changes were analyzed on hematoxylin/eosin stained 5?μm sections of control 7d PI retinas. In control eyes, ERG exhibited a pattern similar to a standard recording. Retinas subjected to mild ischemia preserved ordered layered morphology, exhibiting approximately 30% loss of ganglion cells and no changes in gross morphology. By day 3 PI, ischemia caused an increase in the a-wave amplitude (from 34.9 ± 2.7 to 45.4 ± 4.3 μV), a decrease in the b-wave amplitude (from 248 ± 13 to 162 ± 8 μV), an increase in a-wave latency (from 11.1 ± 0.3 to 17.3 ± 1.4 ms) and b-wave implicit time (from 81.0 ± 1.6 to 90.0 ± 2.5 ms), and attenuation of OPs. The described setup proved sensitive and reliable for evaluating subtle changes in the retinal function in small animals.     

Haem oxygenase-1 gene transfer protects retinal ganglion cells from ischaemia/reperfusion injury

RGC (retinal ganglion cell) death following ischaemic insult is the major cause of a number of vision-threatening diseases, including glaucoma. The aim of the present study was to evaluate the role of HO-1 (haem oxygenase-1) in the retina against IR (ischaemia/reperfusion) injury. Adenovirus-mediated HO-1 gene transfer (Adv-HO-1) was carried out by injection into the vitreous body to induce HO-1 overexpression. At 3 weeks after transfection, levels of HO-1 expression, as measured by Western blot analysis, immunohistochemical staining and activity assay, were drastically up-regulated. Transient retinal ischaemia was induced by raising the intraocular pressure to 150 mmHg for 60 min. Untreated IR caused a significant decrease in RGC numbers at 3 and 7 days after reperfusion (76.1 and 67.2% of control eyes with sham IR respectively; P<0.001). Eyes pretreated with Adv-HO-1 had less RGC loss on day 3 and 7 following reperfusion compared with control eyes injected with Adv-GFP (adenovirus containing a gene for green fluorescent protein; 94.3 and 88.2% respectively; P=0.007 and 0.001). SnP (tin protoporphyrin), an HO-1 inhibitor, counteracted the effects of Adv-HO-1. In conclusion, these findings provide evidence that augmentation of HO-1 enzyme overexpression by intravitreal injection is able to protect RGCs against IR-induced damage.     

Delivery of ciliary neurotrophic factor via lentiviral-mediated transfer protects axotomized retinal ganglion cells for an extended period of time

Ciliary neurotrophic factor (CNTF) has recently been demonstrated to be one of the most promising neurotrophic factors to improve both the survival and regeneration of injured retinal ganglion cells (RGCs). In the present study, we used optic nerve transection as an in vivo model to evaluate the effectiveness of a self-inactivating, replication-deficient lentiviral-mediated transfer of human ciliary neurotrophic factor (SIN-PGK-CNTF) on the survival of axotomized adult rat RGCs. Counts of dextran-fluorescein isothiocyanate conjugated (D-FITC)-retrogradely labeled RGCs revealed that the percentage of RGCs was drastically reduced (<90% cell death) 21 days after optic nerve transection. Retinal sections stained with X-gal revealed that intravitreal injection of the control LacZ-expressing lentiviral vector (LV-LacZ) resulted in the transduction of RGCs and retinal pigment epithelium (RPE) cells. A single intravitreal injection of LV-CNTF at the time of axotomy significantly enhanced RGC survival at 14 and 21 days postaxotomy compared to controls. These results demonstrate for the first time that rapid and prolonged delivery of CNTF using lentiviral-mediated gene transfer to the retina is an effective treatment for rescuing axotomized RGCs for an extended period of time. These results suggest that early and continuous administration of CNTF could serve as a potential treatment for retinal disorders involving optic neuropathy and RGC injury such as in glaucoma.