绿色荧光蛋白逆转录病毒及其介导的视网膜色素上皮细胞基因转移研究

目的 制备携带绿色荧光蛋白（green fluorescent protein,GFP)基因的逆转录病毒,感染原代与建株的人视网膜色素上皮（retinal pigment epithelium,RPE)细胞,评价逆转录病毒对人RPE细胞的感染能力,为视网膜病变的基因治疗奠定基础。方法 分离编码GFP的cDNA片段并插入逆转录病毒载体pLNCX,借助脂质体将重组逆转录病毒载体pLNCX-GFP转入单嗜性及双嗜性包装细胞,G418筛选抗性克隆,分离GFP表达水平最高的克隆;收集含病毒颗粒的上清液感染NIH3T3及体外培养的人原代与建株的RPE细胞。结果 重组逆转录病毒载体pLNCX-GFP转染各种包装细胞后,可产生GFP逆转录病毒。由双嗜性包装细胞产生的GFP逆转病毒能够感染原代与建株的人RPE细胞,GFP基因可持续在RPE细胞内表达。结论 逆转录病毒能够简便、快速、稳定将目的基因转入RPE细胞,可作为眼底病变基因治疗介导目的基因转移的重要工具。     

含抗凋亡基因bcl-2重组逆转录病毒重组体的构建及鉴定

目的:构建并鉴定含鼠bcl-2基因重组逆转录病毒。方法:将抗凋亡基因bcl-2片段从载体pcDNA3.1中切下,定向插入逆转录病毒载体PLNCX2中,酶切鉴定;脂质体法将重组逆转录病毒转入包装细胞系PT67,G418筛选建立稳定表达bcl-2的细胞株。结果:双酶切鉴定,成功构建重组逆转录病毒载体;重组逆转录病毒转入包装细胞PT67,经G418筛选,形成了抗性克隆,并测得病毒滴度为3×1011cfu/L,提示构建的含鼠bcl-2基因重组逆转录病毒载体及稳定的包装细胞系成功。结论:含抗凋亡基因bcl-2重组逆转录病毒载体构建成功。     

逆转录病毒载体携带报告基因在色素上皮细胞的转染与表达

目的 观察逆转录病毒载体携带报告基因在视网膜色素上皮(retinal pigment epithelium,RPE)细胞的表达情况,以探讨基因疗法用于抑制增生性玻璃体视网膜病变的可行性.方法 选第3～4代培养的已长满融合的RPE细胞,以1:3的分种率分种传代.随机分为对照组与实验组,每组12孔.实验组用绿色荧光蛋白基因作为报告基因,以逆转录病毒载体携带来转染RPE细胞,重组合绿色荧光蛋白基因的逆转录病毒上清液浓度为1.2×109 cfu·L-1.对照组RPE细胞以同样方法直接转染绿色荧光蛋白基因质粒DNA.观察绿色荧光蛋白基因在2组RPE细胞的表达情况.结果 基因转染后可见实验组RPE细胞的绿色荧光蛋白基因表达率可达56%～72%,对照组未见有任何表达.结论 逆转录病毒载体可以携带目的基因转染于RPE细胞,并达到较高的转染率.     

疱疹性口腔炎病毒G蛋白/胸苷激酶逆转录病毒载体转移人视网膜色素上皮细胞及其表达

目的　应用新型逆转录病毒载体介导单纯疱疹病毒胸苷激酶 (herpessimplexvirus thymidinekinase ,HSV TK)基因转移 ,探讨丙氧鸟苷 (ganciclovir,GCV)对HSV TK基因修饰人视网膜色素上皮细胞 (retinalpigmentepithelialcells,RPE)的杀伤抑制作用。方法　生产制备高滴度疱疹性口腔炎病毒G蛋白 (vesicularstomatitisvirus Gprotein ,VSV G) /胸苷激酶 (thymidinekinase ,TK)和VSV G/LacZ病毒 ,对NIH3T3细胞进行VSV G/TK滴度测定 ;VSV G/LacZ感染CRL2 30 2细胞后 ,以X gal染色估计感染率 ;对CRL2 30 2细胞、分离培养的RPE细胞及NIH3T3细胞感染不同病毒滴度的VSV G/TK ,结合GCV作用 ,观察 3种细胞生长抑制率。结果　VSV G/TK滴度为 1 2× 10 8克隆形成单位 /ml;VSV G/LacZ感染CRL2 30 2细胞的X gal染色蓝染细胞率为 5 8% ;当感染复数为 2 0 0时可最大的抑制细胞生长 ,抑制率为 4 5 %。结论　VSV G逆转录病毒可以介导LacZ、HSV TK基因在离体NIH3T3细胞和人RPE细胞中高效转移和表达。被HSV TK基因修饰的细胞对GCV作用敏感 ,其生长受到抑制。     

αA晶状体蛋白重组腺病毒载体的构建和鉴定

目的构建携带鼠αA晶状体蛋白的腺病毒表达载体,为进一步研究αA晶状体蛋白高表达对损伤条件下神经细胞的保护作用提供实验基础。方法采用RT-PCR扩增的方法获取鼠源性的αA晶状体蛋白基因,并克隆入穿梭质粒pAdTrack-CMV,构建携带外源性αA晶状体蛋白编码基因的重组腺病毒载体pAd-CRYAA。脂质体法转染人胚肾293细胞,包装产生重组腺病毒Ad-CRYAA。应用PCR鉴定重组腺病毒,空斑传代纯化病毒并反复冻融扩增病毒。以腺病毒感染滴度快速测定试剂盒测定病毒滴度。结果 PCR、序列测定以及限制性酶切证实αA晶状体蛋白基因正确插入腺病毒表达载体,并在人胚肾293细胞中包装出重组腺病毒。收获病毒的滴度为1.143×1012ifu·L-1。结论成功构建Ad-CRYAA重组腺病毒表达载体,可作为后续研究中可靠的基因转染工具。     

HTRA1 shRNA慢病毒表达载体的构建及感染RPE细胞株的鉴定

 目的 构建HTRA1基因的shRNA慢病毒表达载体,并鉴定HTRA1 shRNA慢病毒感染RPE细胞株的效果。设计 实验研究。研究对象 HTRA1 shRNA慢病毒载体和RPE细胞。方法 设计靶向HTRA1 mRNA的寡核苷酸序列,构建HTRA1 shRNA表达质粒,测序鉴定其序列的正确性。通过载体质粒pGC-LV与辅助质粒pHelper 1.0、pHelper 2.0共转染293T细胞包装慢病毒,收集病毒上清,测定滴度。将包装好的HTRA1 shRNA慢病毒感染RPE细胞,设阴性对照和空白对照,采用实时PCR（RT-PCR）和蛋白印迹法（Western Blot）方法检测HTRA1基因的mRNA水平和蛋白质表达水平的变化。主要指标  HTRA1基因的mRNA和蛋白质表达量。结果 DNA测序证实HTRA1 shRNA表达质粒包装了正确的RNA干扰序列。慢病毒滴度测定为8×108 TU/ml。HTRA1 shRNA慢病毒感染RPE细胞后,HTRA1基因的mRNA水平和蛋白质表达水平较空白对照组和阴性对照组均明显下降,差异均有统计学意义。结论 成功构建了HTRA1 shRNA的慢病毒表达载体,该shRNA慢病毒能够有效地抑制RPE细胞株HTRA1基因的表达。     

基因治疗对Leber先天性黑矇视功能的影响

RPE65基因突变引起的早发性、严重视网膜营养不良常合并先天弱视和成年初期视力完全丧失,该基因的编码蛋白是一种相对分子质量为65 KD的视网膜上皮特异蛋白。我们在3例年轻患者视网膜下注射重组腺病毒载体2/2,载体可在人视网膜RPE65基因启动子控制下表达RPE65反转录DNA(cDNA)。     

miR-29b激活自噬抑制人视网膜色素上皮细胞增殖的实验研究

目的　研究miR-29b激活自噬抑制人视网膜色素上皮（retinal pigment epithelial，RPE）细胞增殖的作用。方法　通过病毒颗粒包装转染RPE细胞24~72 h，在倒置荧光显微镜下观察荧光强度检测感染效率，利用实时定量PCR检测miR-29b mRNA表达。采用凝血酶刺激RPE细胞增殖模拟增生性玻璃体视网膜病变模型。使用0.5×10³ U·L^-1凝血酶预刺激1 h，然后分别加入miR-29b和Lenti-vector空载体刺激24 h。分为空白对照组（Sham组）、凝血酶刺激组（RPE组）、凝血酶刺激组+miR-29b过表达组（RPE+Lenti-29b组）、凝血酶刺激组+空载体组（RPE+Lenti-vector组）。利用MTT微量酶比色法检测各组RPE细胞增殖情况，Western blot观察微管相关蛋白1轻链3（light chain 3,LC3）Ⅱ表达变化。结果　在倒置荧光显微镜下观察发现，RPE+Lenti-29b组和RPE+Lenti-vector组90%以上的RPE细胞成功转染，且RPE+Lenti-29b组miR-29 mRNA水平高表达，与其余3组相比差异均有统计学意义（均为P<0.01）。与Sham组比较，其余3组RPE细胞增殖明显，差异均有统计学意义（均为P<0.05），LC3Ⅱ蛋白表达也明显升高，差异均有统计学意义（均为P<0.05）；与RPE+Lenti-29b组比较，RPE组和RPE+Lenti-vector组细胞增殖明显（均为P<0.05），但LC3Ⅱ蛋白表达明显低于RPE+Lenti-29b组，差异有统计学意义（P<0.01）。结论　上调miR-29b表达可激活RPE细胞中LC3Ⅱ蛋白表达，提高细胞自噬水平，抑制RPE细胞增殖，可能在增生性玻璃体视网膜病变发生及发展中扮演重要作用。     

HTLV-1 Tax 基因真核表达载体的构建及对 RPE 细胞的影响

目的构建人T淋巴细胞白血病病毒-1(human T-cell leukemia virus type1,HTLV-1)Tax基因真核表达载体,在人视网膜色素上皮(retinal pigment epithelium,RPE)细胞表达Tax蛋白,观察其对细胞的影响。方法应用基因重组技术构建重组载体PIRES2-EGFP-Tax(PT),鉴定后将重组载体转染入RPE细胞,应用RT-PCR、Western-blot和免疫荧光法检测细胞内Tax基因和蛋白的表达;通过MTT和流式细胞仪检测质粒(空白载体、PT和Green-Tax)转染RPE细胞24h和48h后对RPE细胞生长活力及细胞周期的影响。结果经双酶切鉴定和测序分析证实成功地构建了HTLV-1Tax真核表达载体;通过RT-PCR、Western-blot和免疫荧光法检测到转染Tax重组载体的RPE细胞中有Tax基因和蛋白的表达。质粒转染RPE细胞后,细胞生长活力受到抑制,G1期减少而S期增加。但转染Tax质粒和空白载体,对RPE细胞的生长活力和细胞周期产生相似影响。结论成功构建了HTLV-1Tax基因真核表达载体,并在RPE细胞中表达Tax蛋白;Tax质粒对RPE细胞的影响不排除是转染试剂所致。     

抗凋亡基因bcl-XL转染视网膜感光细胞的研究

目的 :探讨介导抗凋亡基因bcl-XL 转染视网膜感光细胞的有效方法。方法 :采用视网膜神经胶质细胞条件培养液体外培养的视网膜感光细胞 ;用脂质体LIPOFECTAMINETM2 0 0 0介导 pEGFP -C3 -bcl-XL 转染至感光细胞 ;采用滴度为 6 5× 10 12 pfu/L的重组腺病毒rAd -gfp -bcl-XL 转染感光细胞 ;在荧光显微镜下观察感光细胞中的绿色荧光蛋白表达情况来比较脂质体、重组腺病毒转染法的转染率 ;用免疫组化比较这两种方法转染的细胞中Bcl-XL蛋白水平。结果 :脂质体、重组腺病毒转染法的转染率分别为 0 1%、 60 % ,免疫组化示重组腺病毒转染的细胞的Bcl -XL 蛋白水平较脂质体转染的细胞高。结论 :在视网膜感光细胞的基因转染中 ,腺病毒介导的目的基因bcl-XL 的转染率及表达明显高过脂质体法 ,重组腺病毒介导的基因转染法是视网膜变性性疾病基因治疗的一种较理想方法。     

Ability of retroviral transduction to modify the angiogenic characteristics of RPE cells

Background: Retinal pigment epithelial (RPE) cells play an important role in the modulation of ocular angiogenesis. Transduction of RPE cells with retroviral vectors bearing modulating genes can result in long-term transgene expression and may alter the angiogenic characteristics of RPE cells. This study was designed to determine whether changes in angiogenic characteristics of RPE cells result from transduction with retroviral vectors bearing modulating genes, using in vitro angiogenic assays, including analysis of endothelial proliferation and wound healing. • Methods: Human RPE cells were transduced with retroviral vectors bearing either a urokinase-type plasminogen activator (u-PA) or a tissue-type plasminogen activator (t-PA) cDNA. Ten weeks after gene transfer, RPE cells transduced with the u-PA (u-PA-RPE cells) or the t-PA cDNA (t-PA-RPE cells), or untransduced (control) RPE cells, were cocultured with human umbilical vein endothelial cells (HUVECs) by contacting and non-contacting coculture methods. The effects of these cells on proliferation and in vitro “wound healing” of HUVECs were evaluated. • Results: Over 18 weeks, u-PA-RPE cells released large amounts of biologically active u-PA (total amount, 50.2 ± 9.7 ng/10⁶ cells/24 h), while t-PA-RPE cells released large amounts of functional t-PA (15.4 ± 3.2 ng/10⁶ cells/24 h). Control RPE cells did not release any detectable t-PA or u-PA. In the proliferation assay, u-PA-RPE cells stimulated HUVEC proliferation in contacting cell cultures, but not in non-contacting cell cultures. In contrast, t-PA-RPE cells, normal RPE cells or exogenous u-PA had no effect on HUVEC proliferation. In the wound healing assay, u-PA-RPE cells in contacting coculture and exogenous u-PA stimulated wound healing of HUVECs, while non-contacting u-PA-RPE cells, t-PA-RPE cells and normal RPE cells had no effect on HUVEC wound healing. RPE cells transduced with u-PA secreted large amounts of u-PA for as long as 18 weeks, and these cells stimulate HUVEC proliferation and in vitro wound healing. As a result, the angiogenic characteristics of RPE cells can undergo long-term changes. • Conclusions: These results suggest that genetically modified RPE cells can be used to modulate ocular angiogenesis and may have potential for gene therapy of ocular diseases. Received: 3 January 1997 Revised version received: 7 May 1997 Accepted: 26 May 1997     

Tracking RPE transplants labeled by retroviral gene transfer with green fluorescent protein.

PURPOSE: To determine whether human retinal pigment epithelium (RPE) can be modified by retroviral-mediated gene transfer and to monitor the human RPE cells in the subretinal space of living rabbits with scanning laser ophthalmoscopy (SLO). METHODS: Cultured human fetal retinal pigment epithelium (HFRPE) was exposed to green fluorescent protein (GFP)-transducing retroviral vectors, Moloney murine leukemia virus, and lentivirus. The cultured cells were followed by fluorescence microscopy. Suspensions of GFP-expressing HFRPE were transplanted into the subretinal space of pigmented rabbits, and the transplant sites were examined by SLO for fluorescence, including fluorescein and indocyanine green angiography. The rabbits were euthanatized at different times after transplantation, and the retinas were studied histologically. RESULTS: Retroviral gene transfer can introduce a foreign gene such as GFP into cultured HFRPE. Gene expression is maintained in cultured RPE for at least 3 months. The lentiviral vector transduced both nondividing and dividing cells; the Moloney vector only transduced the latter. GFP-expressing cells can be followed in the living retina. Their changes reflect the rejection response followed histologically. CONCLUSIONS: Cultured HFRPE could be transduced to express GFP for long periods of time by retroviral gene transfer. GFP allowed retinal transplants and gene expression to be monitored in vivo. These results provide a model for potential ex vivo gene therapy in the subretinal space.     

Long-term real-time monitoring of adeno-associated virus-mediated gene expression in the rat retina

Purpose : Previous studies have demonstrated that adeno‐associated virus (AAV) efficiently transduced retinal pigmented epithelial (RPE) cells. The goal of this study was to further evaluate and characterize transgene expression within the RPE cells over time in vivo. Methods : Adeno‐associated virus‐mediated gene transfer was monitored and quantified by retinal photography following subretinal injection of a recombinant AAV encoding the green fluorescent protein gene (rAAVCMV‐gfp) into rat eyes. Retinal function of transduced rat eyes was measured by electroretinography. Results : The maximum level of transgene expression was reached at 8 weeks postinjection followed by a gradual decrease throughout the experimental period. Interestingly, it was observed that while gfp expression was stable in some RPE cells, gfp fluorescence completely disappeared in other cells over the duration of the experiment. The expression of AAV‐mediated gfp in RPE cells did not alter the retinal function for over 1 year. Conclusions : These results confirm the importance of this direct visualization system to study vector transgene expression in vivo and support the use of AAV for diseases treatable by targeting RPE cells. Key words : adeno‐associated virus, gene transfer, green fluorescent protein, retinal photography, retinal pigmented epithelium.     

The possibility of gene therapy for the treatment of choroidal neovascularization

PURPOSE: Choroidal neovascularization (CNV) is responsible for most cases of severe visual loss in age-related macular degeneration. Recently, the possibility of gene therapy has been proposed for the treatment of CNV. The purpose of this study was to examine the feasibility of ex vivo and in situ gene therapy approaches for CNV. DESIGN: Experimental study. METHODS: Human retinal pigment epithelial (RPE) cells were transduced with a retroviral vector coding for beta-galactosidase. Transduced cells were grown on type II collagen sheets and transplanted under the retina of 20 rabbits. Animals were observed for 3 to 56 days, and transplanted cells were examined histologically and with X-gal staining. Bovine choroidal endothelial cells (CEC) were transduced with retroviral vectors coding for tissue inhibitor of metalloproteinase-2 (TIMP-2) or control vector. Production of TIMP-2 by transduced cells was determined by immunohistochemical analysis and enzyme-linked immunosorbent assay. Effect of transduction on in vitro proliferation, migration, and tube formation was examined in response to vascular endothelial growth factor (VEGF). Four CNV lesions were induced in one cynomolgus monkey by laser photocoagulation. Two days later, retroviral vector coding for TIMP-2 or control vector was injected into the subretinal space overlying the CNV lesions. The monkey was observed for 12 weeks using fluorescein angiography. RESULTS: Transplantation of transduced RPE cells was technically achieved in 10 of 20 animals. In these animals, RPE cells at the site of transplantation formed a monolayer and expressed beta-galactosidase for 14 days. beta-Galactosidase-positive cells were not identified at 56 days. Choroidal endothelial cells transduced with TIMP-2 secrete TIMP-2 into the media and show decreased migration and tube formation in vitro. In the in vivo monkey model, the control CNV lesions (n = 2) showed prominent leakage, whereas the experimental lesions (n = 2) showed minimal hyperfluorescence. CONCLUSIONS: Retrovirally transduced RPE cells survive in the subretinal space for at least 14 days and continue to express the gene product coded for by the vector. Choroidal endothelial cells retrovirally transduced for TIMP-2 produce TIMP-2 in vitro and show decreased angiogenic responses in vitro in response to VEGF. A preliminary study attempting in situ delivery of TIMP-2 vector to CNV lesions in a monkey eye supports the feasibility of this approach and encourages further study.     

Angiopoietin‐like protein 4 (ANGPTL4) is induced by high glucose in retinal pigment epithelial cells and exhibits potent angiogenic activity on retinal endothelial cells

Purpose: Hyperglycaemia has been identified as major risk factor for diabetic retinopathy (DR). It is widely accepted that the progression of DR is mainly due to a local imbalance of pro‐ versus anti‐angiogenic factors in the retina. In this study, we investigated whether retinal pigment epithelial (RPE) cells produced pro‐angiogenic factors under high glucose (HG) conditions in vitro. Methods: Cultured human retinal endothelial (RE) cells were exposed to conditioned medium from retinal pigment epithelium cells (ARPE‐19) grown in HG medium and assessed for tube formation. Based on the expression profiles of ARPE‐19, we investigated whether ANGPTL4 was a major angiogenic factor released from ARPE‐19 under HG conditions using cultured human RE cells as the test system for experiments with recombinant protein, conditioned medium from ARPE‐19 and RNA interference (RNAi). Results: The conditioned medium from ARPE‐19 cultured under HG conditions promoted tube formation of cultured human RE cells. GeneChip analysis showed that ANGPTL4 was one of the highest upregulated genes under HG conditions. In addition, recombinant ANGPTL4 promoted all of the elements of angiogenesis in human RE cells in vitro. The results of experiments using conditioned medium from ARPE‐19 combined with RNAi demonstrated that ANGPTL4 was a major angiogenic factor released from ARPE‐19 under HG conditions. Conclusions: ANGPTL4 was induced by high glucose in RPE cells and exhibited potent angiogenic activity on RE cells. Our results are unique and may potentially add a new candidate to the long list of molecules involved in diabetic retinopathy.     

Transplantation of transduced retinal pigment epithelium in rats

PURPOSE: To examine the effects of transplanting retinal pigment epithelial (RPE) cells transduced with neurotrophic factor genes into the subretinal space of rats. METHODS: RPE cells were transduced with plasmids carrying the cDNAs of Axokine (ciliary neurotrophic factor [CNTF]; Sumitomo Pharmaceuticals Co., Ltd., Tokyo, Japan), brain derived-neurotrophic factor (BDNF), and basic fibroblast growth factor (bFGF) genes. These RPE cells were transplanted into the subretinal space of rats, and the localization was examined. The expression of enhanced green fluorescent protein (eGFP)-BDNF-transduced RPE in the subretinal space was examined by real-time polymerase chain reaction (PCR) after the transplanted cells were collected by cell sorting. The expression of major histocompatibility complex (MHC) class I and -II after gene transduction was examined by real-time PCR. The ratio of CD4(+) and CD8(+) T cells and antibody production against transplanted cells were analyzed by flow cytometry. RESULTS: The transplant sites were not significantly different among the neurotrophic factors tested. The RPE cells expressed the BDNF gene in the subretinal region at approximately the same level as that in vitro. RPE cells transduced with Axokine stimulated MHC-I expression, and the cell transplantation changed the ratio of CD4(+) and CD8(+) T cells. A significant production of antibody against the Axokine-transduced RPE cells was also observed after Axokine-transduced RPE transplantation. CONCLUSIONS: RPE cells transduced with neurotrophic factors express the factors after transplantation into the subretinal space. RPE transduced with Axokine or bFGF, in contrast to RPE transduced with BDNF, stimulate an immunologic reaction of the host.     

A novel serum-free method for culturing human prenatal retinal pigment epithelial cells

PURPOSE: Established techniques for culturing primary human retinal pigment epithelial (RPE) cells have facilitated the laboratory investigation of this multipurpose retinal cell layer. However, most culture methods involve the use of animal serum to establish and maintain RPE monolayers, which can complicate efforts to define and study factors involved in the maturation and function of these cells. Therefore, this study was conducted to develop a simple, serum-free system to propagate and sustain human RPE in vitro. METHODS: RPE was dissected from human prenatal donor eyes and cultured in serum-free defined medium containing the commercially formulated supplement B27 or N2. Cultures were grown initially as adherent tissue sections or suspended spherical aggregates and later expanded and maintained as monolayers. PCR, Western blot analysis, and immunocytochemistry were used to monitor gene and protein expression in established cultures, followed by examination of secretory products in RPE conditioned medium by ELISA and mass spectrometric analysis. RESULTS: In medium supplemented with B27, but not N2, RPE could be expanded up to 40,000-fold over six passages and maintained in culture for more than 1 year. In long-term cultures, typical cellular morphology and pigmentation were observed, along with expression of characteristic RPE markers. RPE monolayers also retained proper apical-basal orientation and secreted multiple factors implicated in the maintenance of photoreceptor health and the pathogenesis of age-related macular degeneration. CONCLUSIONS: Monolayer cultures of human prenatal RPE can be grown and maintained long term in the total absence of serum and still retain the phenotype, gene and protein expression profile, and secretory capacity exhibited by mature RPE cells.     

Recombinant Sendai virus-mediated gene transfer into adult rat retinal tissue: efficient gene transfer by brief exposure

To determine the usefulness of recombinant Sendai virus (SeV) for ocular gene transfer, the authors characterized SeV-mediated gene transfer to the retinal tissue of adult rats via subretinal injection. Recombinant SeV encoding the lacZ gene achieved frequent transgene expression in the retinal pigment epithelium (RPE) (mean=38.76%), while gene transfer to other retinal cells was rare. These findings are similar to those of previous reports using adenoviruses. Peak reporter gene expression of SeV in cultured RPE cells was similar to that of adenovirus at the same titer; however, SeV achieved high levels of expression after a brief vector-cell contact time, while adenovirus required over 3hr for efficient gene transfer. This finding was also observed in vivo following a brief SeV filling in the subretinal space, and may therefore provide a clinical advantage in avoiding retinal damage due to prolonged detachment. The observed SeV-mediated gene expression in the rat retina was transient. The initial phase of the decrease in luciferase activity could be prevented by daily eye drops of dexamethasone, suggesting that the corticosteroid-sensitive host reaction may affect early clearance of the virus. The late decline of transgene expression (2 weeks) was inhibited by the immunosuppressant, cyclosporin A, in a dose-dependent manner, suggesting that the cytotoxic T-lymphocyte response may be important in this phase. This work represents the first report of SeV-mediated gene transfer to ocular tissue, and identifies recombinant SeV as a new tool for studies of retinal gene transfer and gene therapy.