X-linked retinoschisis (XLRS) is a rare X-linked inherited retinal disorder, caused by mutations in retinoschisin 1 (RS1) gene. Three XLRS mice were established, providing ideal systems to study the mechanism and treatment methods for XLRS. RS1 gene mutations can induce abnormal secretion or adhesion function of RS1 protein. In the past year, phase I clinical trials for XLRS has begun in USA, using adeno associated virus (AAV, AAV8 or AAV2)-mediated gene delivery. With the rapid development of new generation of AAV vector that can transduce more retinal cells through intravitreous delivery, gene therapy for XLRS will have a brighter future.
Objective To observe the permeability and stability of the transfection of antisense oligonucleotide (ASODN) hybridized epidermal growth factor receptor (EGFR) to retinal glial cells (RG).Methods Phosphorothioate and unmodified EGFR ASODN conjugated with 5′-isothioc yanate (5′-FITC) were encapsulated with or without lipofectin, and then added into human retinal glial cells culture media. The cellular permeability and stability of the transfection were observed by fluorescence microscopy in fixed cells.Results In the absence of lipofectin, phosphorothioate and unmodified EGFR ASODN were found in a few RG cells at 30 minutes, and in about 50% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in RG cells for 3-4 hours and disappeared at about 8 hours. In the presence of lipofectin, phosphoro thioate and unmodified EGFR ASODN were found in a few RG cells at 15 minutes and about 70%-80% RG cells at 4 hours. Phosphorothioate EGFR ASODN were kept in cells for 10-12 hours, and phosphorothioate and unmodified EGFR ASODN were disapp eared at about 14 hours and 4 hours respectively.Conclusion 5′-FITC EGFR ASODN encapsulated with lipofectin could enter RG cells and express stably in RG cells. (Chin J Ocul Fundus Dis，2003，19：52-54)
Objective To invesitgate the influence of recombinant adenovirus vector of human pigment epithelium-derived factor（AV-hPEDF）on retinal new vessels mediated by recombinant adenovirus vector. Methods Twenty 7-days-old Sprague-Dawley (SD) rat were divided into two groups randomly after the establishment of retinal neovascularization model. At postnatal 14 day, they were accepted intravitreal injection with blankadenovirus-vector (AV-Blank group) and adenovirus-vector PEDF(AV-PEDF group) respectively. The retinal vascular endothelial cells were counted, the PEDF mRNA and protein expression in retina and vitreous were determined by reverse transcriptionpolymerase chain reaction (RT-PCR) and immunohistochemistry. Results After injection with medicine, the number of RNV was decreased obviously in AV-PEDF group（t＝42.009，Plt;0.001）;the protein expression of retinal PEDF was increased obviously in AV-PEDF group（t=36.638，Plt;0.001）; the PEDF mRNA expression in vitreous was also increased obviously in AV-PEDF group （t=9.128，Plt;0.001）. Conclusion Recombinant Adenovirus vector mediated PEDF can raise the PEDF expression in the retinal and vitreous neovascularized tissues in rat, which suggested that the expression of PEDF may be related to inhibition and reduction of RNV.
The application of gene therapy in ocular diseases is gradually expanding from mono-gene inherited diseases to multigene, multifactorial, common and chronic diseases. This emerging therapeutic approach is still in the early exploratory stage of treating diseases, and the expected benefits and risks remain highly uncertain. In the delivery process of gene therapy drugs, viral vector is currently one of the most mature and widely used vectors. The occurrence of vector-associated immunity will affect the short-term and long-term effects of gene therapy, and even cause permanent and serious damage to visual function. Therefore, gene therapy vector-associated immunity is the focus and challenge for the safety and long-term efficacy of gene therapy. During the perioperative and follow-up of gene therapy, attention should be paid to the monitoring of vector-associated immune inflammation, and appropriate measures should be taken to deal with the corresponding immune response, so as to achieve the best visual benefits for patients.
Purpose To study the possibility of prevention of proliferative vitreoretinopathy（PVR） by transduction of exogenous gene in vivo． Methods PVR model of rabbits was induced by intravitreal injection of fibroblasts．beta;-galactosidase (lacZ) gene as a reporter gene was transfered into the vitreous of PVR model eyes mediated by retroviral vector, and the expression of the gene in eye tissues was determined ． Gene transfection was done on the 6th day after fibroblasts injection,and the dosage of intravitreal injection of reporter gene was 0．1ml PLXSN/lacZ serum-free supernatant (1．1times;106 cfu/ml)． Results lacZ gene expression was seen in proliferative membranes after gene transfection, and the expression was located maily at the surface of PVR membrane．The reporter gene expression lasted at least more than 30 days．No expression was found in retinal tissues． Conclusions Retrovirus mediated gene can be directionally transducted in PVR membrane,and might possess the feasibility of gene therapy for PVR． (Chin J Ocul Fundus Dis, 2001,17:224-226)
Objective To observe the expression of the pigment epithelium derived growth factor (PEDF) in retina and the effects of PEDF for vascular endothelial growth factor (VEGF) and retinal microvascular after recombinant adeno-associated virus mediated pigment epithelium derived factor（rAAV-mediated-PEDF）transferred retina of diabetic rats. Methods Male Wister rats were induced diabetes with intraperitoneal injection of streptozocin, then divided into 1 month group (DM1), 3month group(DM3) and 6 month group randomly. In diabetic rats, right eyes were injected rAAV2CMVhPEDF into vitreum as treat group,left eyes were injected into rAAV2-CMV-GFP (1times;1011 v.g/ml) as self-control group. In normal rats, right eyes were punctured but not injected (CONf), left eyes let it be without any disposal. The levels of VEGFmRNA and hPEDFmRNA in retina were evaluated using RT-PCR in different period. The protein levels of VEGF, PEDF within the retina were determined using western blot. The change of retinal capillary were observed through retinal vascular flattening. Result The expression of hPEDFmRNA in retina was enhanced persistence after rAAV2-CMV-hPEDF injected, achieved climax until 6 months. The levels of PEDF were also incerased consecutive, the differences were statistically significant in treatment group compared with own control group （Plt;0.01）. Levels of mRNA and protein of VEGF at different time-points among therapy group were not statistically significant, all obviously higher than normal （Plt;0.05），but all lower obviously than respectively own control group at the same timepoint （Plt;0.01）. The morphology of retinal capillary was not different significant with normal rats in 1 month diabetic rats. Morphology changes of therapy groups were less than those of respective own control group in DM3 and DM6. Conclusion Intravitreous injection rAAV2-CMV-hPEDF can increase expression of mRNA and protein of PEDF，alleviate lesion of retinal microvascular in early period of diabetic rats and supress expression of VEGF in retina of diabetic rats.The regulation occur on mRNA level. （Chin J Ocul Fundus Dis，2008，24：259-264）
【Abstract】Objective To study the antitumor activity of HSVtk/CD combinative gene toward human cholangiocarcinoma in vivo. Methods Nude mouse models with transplanted subcutaneous cholangiocarcinoma were constructed and divided into 4 groups randomly, each group had 8 mice. Adenovirus solution free from suicide gene was injected in subcutaneous tumors of each mouse of control group. Adenovirus solution containing cytosine deaminase (CD), thymidine kinase (tk) and HSVtk/CD fusional gene were injected into single suicide gene either HSVtk or CD was transinfected into the tumor cells by injecting viras into subcutaneous tumor of mice of CD gene,tk gene and fused CD and tk gene group respectively. 24 hours after the injection, 5fluorocytosine (5FC) and ganciclovir (GCV) were injected introabdominally in each mouse. Growth of the tumors were monitored.Results Tumor growth of the genetransfection groups was suppressed in different degrees. Compared with the control group, the suppressing rates of the genetransfection groups were 41.2%, 55.7% and 70.0% respectively (P＜0.05). Histological examination showed good tumor growth in the control group, and tumor necrosis in the other 3 groups, particularly obvious in the group transfected with pAd(HSVtk/CD).Conclusion Combinative gene system has a b antitumor effect on cholangiocarcinoma in vivo. But it’s not powerful enough to eliminate tumor thoroughly because of insufficient “Bystander effect”.
The rapid development of genetic diagnosis-related technologies has paved a wide road for gene therapy. Different gene therapy clinical trials for retinal disorders, including gene-replacement therapy, anti-neovascular gene therapy and opotogenetic gene therapy, have been developed and achieved fruitful results, which have gradually confirmed the efficacy and safety of adeno-associated virus (AAV)-mediated gene therapy for recessive retinal diseases. In recent years, novel gene editing technologies also shows great potential to treat dominant retinal disease, or recessive retinal disease when the therapeutic gene fragments are too long to fit into the AAV vectors. These results make it possible for most of the patients with inherited retinal diseases to be treated by the safe and effective AAV-mediated gene therapy, which will also benefit Chinese patients soon.