AAV载体介导的视网膜基因转移实验研究

目的以绿色荧光蛋白(green fluorescent protein,GFP)作为标记基因,观察腺相关病毒(AAV)载体介导视网膜基因转移作用,为视网膜疾病的基因治疗打下基础.方法制备重组腺相关病毒rAAV-gfp;将纯化的rAAV-gfp病毒10μL(10×10     

rAAV载体介导基因对糖尿病大鼠视网膜转染的实验研究

目的以重组腺相关病毒载体(recombinant adeno-associated virus,rAAV)携带色素上皮衍生因子(pigment epithelium-derived factor,PEDF)和绿色荧光蛋白(green fluorescent protein,GFP)基因转染入糖尿病大鼠视网膜,观察其定位及表达情况。方法Wistar大鼠分为正常组(CON)及实验组。实验组以链脲佐菌素诱导形成糖尿病模型后,随机分为1个月组(DM1)、3个月组(DM3)和6个月组(DM6)。右眼玻璃体腔内注射2μLrAAV2-CMV-hPEDF,左眼注射等量rAAV2-CMV-GFP。CON组右眼注射2μL生理盐水。用荧光显微镜观察GFP表达部位,用RT-PCR测定PEDF的表达情况。结果荧光显微镜显示,DM1组视网膜全层,主要是节细胞层表达强荧光。DM3组全层视网膜和部分巩膜表达强荧光。DM6组视网膜及巩膜全层表达强荧光,荧光强度、范围与DM3组相比明显增加。RT-PCR显示,人PEDF mRNA在CON、GFP注射眼组大鼠视网膜均无表达。在右眼实验组1个月时可见hPEDF mRNA表达,并随时间延长,表达不断增强,持续到6个月。结论AAV载体可将PEDF及GFP基因有效地转染入糖尿病大鼠视网膜内并长期表达超过6个月。     

重组腺病毒介导的gfp-bcl-X_L对视网膜变性鼠光感受器细胞保护作用的研究

目的　观察重组腺病毒rAd gfp bcl XL治疗视网膜变性 (retinaldegeneration ,RD)鼠的疗效。方法　 40只RD鼠随机分成A、B组 ,每组 2 0只 ,右眼为治疗眼 ,左眼为对照眼。A、B组治疗眼分别于RD鼠生后 10d及 2 2d视网膜下腔注射含绿色荧光蛋白基因gfp和抗凋亡基因bcl XL 的重组腺病毒颗粒 ;术后 15、30d摘除治疗眼和对照眼行冰冻切片、透射电镜下观察、石蜡切片、HE染色并提取视网膜总RNA ;行逆转录聚合酶链反应分析bcl XL 基因的mRNA表达水平 ;免疫组化分析治疗眼和对照眼的Bcl XL 蛋白水平。结果　治疗眼视网膜有较广泛的绿色荧光蛋白表达 ,说明重组腺病毒成功的将bcl XL 转染至视网膜光感受器细胞 ,且有bcl XL 表达 ;术后 30d ,A组鼠治疗眼和对照眼bcl XL的mRNA表达水平及Bcl XL 蛋白含量有明显差异 ;透射电镜下可见治疗眼的内节段较对照眼完好 ;HE染色示治疗眼视网膜光感受器细胞层较对照眼厚。而B组鼠治疗眼和对照眼的视网膜光感受器细胞层的厚度无明显差异。结论　注射至早期RD鼠视网膜下腔中的rAd gfp bcl XL 能有效转染光感受器细胞并发挥抗凋亡作用 ,但对晚期RD鼠的疗效不明显。     

超声微泡造影剂促进重组腺相关病毒载体介导基因转染视网膜神经节细胞的体内实验

目的 观察超声微泡造影剂提高重组腺相关病毒(rAAV2)介导增强型绿色荧光蛋白(EGFP)基因在体内转染视网膜神经节细胞(RGC)的效率.方法 Sprague-Dawley大鼠40只,随机分为A、B、C、D 4组,每组各10只大鼠.A组玻璃体腔注射磷酸盐缓冲液(PBS)5μl;B组玻璃体腔注射rAAV2-EGFP 5 μl;C组玻璃体腔注射rAAV2-EGFP 5 μl后立即用超声波辐照眼球;D组玻璃体腔注射rAAV2-EGFP和微泡造影剂的混悬液5 μl后立即用超声辐照眼球.玻璃体腔注射21 d后,3%荧光金逆行标记RGC.逆行标记7 d后取出眼球,制作视网膜铺片及视网膜冰冻切片,在激光共聚焦显微镜下观察并计算EGFP基因在RGC的转染率及在RGC表达的平均吸光度[A,旧称光密度(OD)]值;用RGC计数判断损伤情况.结果 荧光金标记RGC后,B、C、D 3组均可观察到RGC中有EGFP表达.其中,D组平均A值为95.02±7.25,RGC转染率为(20.10±0.74)%、均明显高于B、C组,差异有统计学意义(F平均A值=25.970,F转染率=25.799;P<0.01);A、B、C、D组RGC计数差异无统计学意义(F=0.877,P>0.05).结论 在低频和一定能量的超声和微泡造影剂作用下,rAAV2介导EGFP基因转染体内RGC的效率能够安全、有效地提高.     

重组腺相关病毒经不同途径转染鼠眼球组织

目的　研究重组腺相关病毒 (rAAV)介导的报告基因 (LacZ)能否转染到鼠的眼球组织 ;以及rAAV LacZ在眼内转染的最佳途径。方法　 2 0只SD大鼠随机分成 4组 ;第 1组 ,用rAAV LacZ(10 9个病毒颗粒·mL-1)溶液滴眼 ;第 2组 ,结膜囊注射 2 0 μLrAAV LacZ ;第 3组 ,前房注射 10 μLrAAV LacZ ;第 4组 ,玻璃体腔注射 10 μLrAAV LacZ。LacZ基因转染 30d后 ,分别取各组大鼠的眼球行 5 溴 4 氯 3 吲哚 β D 半乳糖苷 (X gal)组织化学染色 ,评价报告基因在眼球的表达情况。结果　X gal组织化学染色证实 ,用rAAV LacZ溶液滴眼 ,仅少量报告基因可以在角膜上皮层表达 ;结膜囊注射rAAV LacZ ,报告基因仅表达于结膜下组织 ;前房注射rAAV LacZ ,报告基因可表达于角膜和虹膜组织 ;玻璃体腔注射rAAV LacZ后 ,报告基因可表达于视网膜组织。结论　rAAV载体能定向携载报告基因到眼内组织 ,rAAV可以用于眼病的基因治疗     

氟尿嘧啶脂质体玻璃体注射防治增生性玻璃体视网膜病变的实验研究

目的 探讨氟尿嘧啶脂质体兔眼玻璃体内注射对增生性玻璃体视网膜病变的防治作用.方法 10只白色家兔,每只眼玻璃体内注射自体腹腔收集巨噬细胞制成增生性玻璃体视网膜病变动物模型.右眼为实验眼,玻璃体内注射氟尿嘧啶脂质体1次,浓度为1.6mg/0.1mL;左眼为空白对照,注射磷酸缓冲液.每周观察玻璃体视网膜病变发展程度.4周观察术后视网膜脱离发生率.结果 术后28天,实验组视网膜脱离发生率为30.00%(3/10),对照组为80.00%(8/10),差异有统计学意义.结论 玻璃体内注射氟尿嘧啶脂质体1次能有效地预防实验性增生性玻璃体视网膜病变的发生.     

重组腺病毒载体介导色素上皮衍生因子基因抑制视网膜新生血管形成

目的 观察重组腺病毒载体介导色素上皮衍生因子（PEDF）基因对视网膜新生血管（RNV）发生的影响。方法 将20只鼠龄7 d 的Spraque-Dawley大鼠建立模型后随机分为2组，分别于出生后14 d 行玻璃体内注射空白腺病毒载体（AV-Blank组）及编码PEDF基因的重组腺病毒载体（AV-PEDF组），采用RNV内皮细胞计数、运用逆转录聚合酶链反应（RT-PCR）和免疫组织化学法检测玻璃体、视网膜中PEDF基因及其蛋白表达的变化。结果 注射药物后AV-PEDF组视网膜的新生血管数较AV-Blank组明显减少（t＝42.009，P＜0.001）；视网膜PEDF蛋白的表达较AV-Blank组明显增加（t=36.638，P＜0.001）；玻璃体组织中的PEDF mRNA表达量也较AV-Blank组明显增加（t=9.128，P＜0.001）。结论 重组腺病毒载体介导的PEDF基因可上调大鼠RNV眼玻璃体及视网膜组织中PEDF的表达；推测PEDF的表达可能与RNV的抑制与消退相关。     

病毒载体介导的视网膜基因转移研究进展

用病毒作为载体携带功能基因转染视网膜以达治疗目的，这种基因治疗方法为某些遗传性视网膜病如视网膜色素变性的治疗开辟了新途径。应用腺病毒或腺相关病毒载体，经视网膜下间隙或玻璃体注射，可使报告基因在视网膜细胞中有效表达。腺病毒介导的基因表达起效早，持续时间短，腺相关病毒介导的基因表达起效晚，持续时间长。病毒载体中所含启动子的不同，其基因导致人的靶组织也不同。含茂细胞病毒启动子的病毒载体可将基因转移至视网膜色素上皮细胞，应用视紫红质启动子则转移至视细胞。     

15-脂氧合酶-1基因转移抑制小鼠视网膜新生血管的实验研究

目的 观察15-脂氧合酶-1(15-LOX-1)基因转移对氧诱导小鼠视网膜新生血管的抑制作用.方法 7日龄C57BL/6J小鼠96只,随机分为正常对照组、氧诱导视网膜病变(OIR)模型组、基因治疗组和空白载体组.将小鼠与哺乳母鼠共同置于氧浓度为(75±2)％的氧箱内饲养5d后转移至正常环境中饲养5d,建立OIR模型.小鼠出生后第12天基因治疗组玻璃体腔注射携带增强型绿色荧光蛋白(EGFP)和小鼠15-LOX-1基因的重组腺病毒(Ad-15-LOX-1-EGFP)载体1μl;空白载体组注射等量携带EGFP的重组腺病毒(Ad-EGFP)载体.注射后第2天行视网膜铺片荧光显微镜观察EGFP的表达.注射后第5天行免疫荧光染色法、实时荧光定量聚合酶链反应和蛋白免疫印迹法检测15-LOX-1基因转染视网膜的表达;视网膜铺片观察视网膜血管变化,测量视网膜无灌注区和新生血管的相对面积;石蜡切片苏木精-伊红染色并计数突破视网膜内界膜的血管内皮细胞核.结果 Ad-15-LOX-1-EGFP注射第2天,视网膜铺片上观察到EGFP的表达.免疫荧光染色结果显示,15-LOX-1基因转染视网膜主要表达在外丛状层、内核层和神经节细胞层.基因治疗组15-LOX-1蛋白和mRNA表达水平明显高于OIR模型组和空白载体组,差异有统计学意义(t蛋白表达水平=22.74、24.13,tmRNA表达水平=12.51、13.40;P＜0.01);基因治疗组视网膜无灌注区和新生血管面积较OIR模型组和空白载体组显著减小,差异有统计学意义(t血管区面积=16.22、14.31,t新生血管面积=9.97、9.07;P＜0.01);基因治疗组中突破视网膜内界膜的血管内皮细胞核与OIR模型组和空白载体组比较明显减少,差异有统计学意义(t=14.25、11.62,P＜0.01).结论 15-LOX-1基因转移不仅可以减少氧诱导小鼠视网膜无灌注区面积,并且对视网膜新生血管有显著的抑制作用.     

内皮抑素基因转移抑制视网膜新生血管的实验研究

目的评价脂质体介导的内皮抑素(ES)基因转移抑制缺氧诱导的小鼠视网膜新生血管的效果。探讨基因转移抑制视网膜新生血管的可行性。方法制备阳离子脂质体及PCDNA3ES复合物。选1周龄C57Bl/6N小鼠置于氧浓度为(75±2)%的氧箱中5d。回到正常环境中诱导视网膜新生血管模型。在小鼠离开氧箱的当日,向ES注射组鼠玻璃体腔注射2μl脂质体PCDNA3ES复合物;载体对照组注射等量脂质体空白载体复合物;空白对照组小鼠注射等量PBS。采用ES抗体免疫组化方法检测ES蛋白在视网膜的表达;回到正常环境中后5d,采用荧光标记的右旋糖酐血管灌注下视网膜铺片方法观察视网膜新生血管的分布;组织学切片观察比较突破视网膜内界膜的血管内皮细胞数量;透射电镜观察ES转移对视网膜超微结构的影响。结果免疫组化检查发现ES注射组小鼠玻璃体腔注射ES后24h开始有ES表达,主要位于视网膜神经纤维层细胞中,维持至少2周仍见表达;视网膜铺片观察可见空白对照组在无灌注区边缘均可见新生血管芽及荧光渗漏。ES注射组见新生血管芽明显减少;组织学检查ES注射组较其他两组突破视网膜内界膜的细胞数量减少,差异有统计学意义;ES转移后电镜下视网膜各层超微结构未见明显改变。结论采用玻璃体腔注射方法行脂质体介导的内皮抑素基因转移可以一定程度抑制缺氧诱导的小鼠视网膜新生血管生长,对视网膜无明显的毒副作用。应进一步优化转移条件以提高治疗效果。     

A feasibility study of recombinant adeno-associated virus as a vector for transferring a target gene to retina

AIM: To study the feasibility of recombinant adeno-associated virus (rAAV) as a vector to transfer the green fluorescent protein (GFP) gene as a target gene into rabbit retina. METHODS: Intravitreal injection of rAAV-gfp was performed in either eye for each rabbit with the other eye taken as control. At the 3rd, 7th, and 14th day after injection, the eyeballs were removed, and the retinas were flat-mounted on glass slides to inspect the retinal fluorescence, respectively. RESULTS: After intravitreal injection of rAAV-gfp, the presence of fluorescent spots in the cytoplasm of retinal cells indicated that GFP gene was efficiently transferred and expressed in the rabbit retina. CONCLUSION: Recombinant adeno-associated virus is a reliable and simple vector for transferring target gene, e.g., GFP gene, to the retina.     

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.     

Cone-specific expression using a human red opsin promoter in recombinant AAV

PURPOSE: To determine the feasibility of targeting gene expression specifically to cone photoreceptors using recombinant adeno-associated virus (rAAV) as the vector. METHODS: An rAAV vector was constructed that contains a 2.1kb upstream sequence of the human red opsin gene to direct green fluorescent protein (GFP) expression. A control construct containing a 472bp mouse rod opsin promoter, previously shown to drive photoreceptor-specific expression, was also used. Each recombinant virus was injected into the subretinal space of rat, ferret or guinea pig eyes. GFP expression was analyzed 4-6 weeks after injection microscopically. RESULT: The human 2.1kb cone opsin gene upstream sequence targeted GFP expression only to a subset of photoreceptors. Cone-specific expression was shown by co-localization of GFP fluorescence and cone-specific opsin antibody staining. Additionally, in rats, expression was specific for L/M-cones whereas no S-cones exhibited GFP fluorescence. The efficiency of rAAV mediated cone transduction surrounding the injection site was high since every L/M-cone antibody-staining cone was also positive for GFP expression. CONCLUSION: The human red/green opsin gene promoter used in this study is sufficient to direct efficient cone-specific gene expression in several mammalian species, suggesting that key cell-type specific regulatory elements must be broadly conserved in mammals. These observations have significance in devising gene therapy strategies for retinal dystrophies that primarily affect cones and point toward a way to functionally dissect the cone opsin promoter in vivo.     

Development and evaluation of the specificity of a cathepsin D proximal promoter in the eye

PURPOSE: Recombinant adeno-associated virus (rAAV)-mediated gene delivery has emerged as a valuable tool for alternative treatment of ocular diseases. Cellular specificity of transgene expression could be influenced by either the viral capsid or the choice of promoter. The use of cellular promoter, cathepsin D (CatD) proximal promoter, and its potential for application in rAAV-based gene therapy are evaluated in this study. MATERIALS AND METHODS: Different sizes of CatD proximal promoter fragments -769 to -1 (CD768), -366 to -1 (CD365), -253 to -1 (CD252), and -124 to -1 (CD123) were subcloned upstream of the green fluorescent protein (GFP) gene. The specificity and activity of the promoter were tested in vitro using human retinal pigment epithelium (RPE) cell lines (RPE51, D407), with the human fibroblast cell line (F2000) used as control. The promoter fragment that showed higher activity in RPE cells was chosen to generate rAAV vector based on AAV serotype 2. The ability of CatD promoter to target transgene expression to RPE in vivo was determined following subretinal delivery of rAAV particles into nonpigmented RCS/rdy+ rats. RESULTS: In vitro studies showed that the proximal promoter fragment CD365 targeted high GFP expression in RPE cells. This fragment was then used to generate the AAV.CD365.gfp construct. It was shown in vivo that following subretinal injection, the CD365 fragment in AAV.CD365.gfp directed GFP expression preferentially into RPE cells. Relatively lower level of GFP expression was detected in the neuroretina. In contrast, injection of control virus (AAV.CMV.gfp) resulted in equal levels of transduction and fluorescence signal intensity in both the RPE and photoreceptor cells. CONCLUSIONS: The results of our study demonstrate that the promoter fragment CD365 has the potential to target preferential gene expression in the RPE following subretinal injection in rAAV-mediated gene therapy.     

腺伴随病毒载体介导Kringle5-GFP基因在眼组织中的表达

目的：探寻腺伴随病毒载体介导Kringle5基因注入玻璃体腔后,在眼球组织中的分布规律,为基因治疗视网膜病变提供实验依据。 方法：将已经构建包装好的rAAV-Kringle5-GFP,滴度为2.5×10¹² vg/mL 10μL注入SD大鼠玻璃体腔后,分别在1,5,10,20,40,60,90d处死SD大鼠,在荧光显微镜下观察GFP在玻璃体、视网膜、脉络膜、虹膜等眼组织分布及表达情况。根据表达强度从无表达到强表达分别记为-,+,++,+++,++++。 结果：在注射后第1d,玻璃体中有轻度表达,随着时间的推移表达缓慢增强,分布较广泛;在玻璃体注射后第5d视网膜有表达,分布范围从视盘到周边部视网膜组织均出现且随着时间的推移表达增强;在虹膜、脉络膜组织表达也有较强表达;甚至在角膜内皮及晶状体组织也有表达。 结论：选用腺伴随病毒载体介导目的基因能够在视网膜组织和色素膜组织有较强表达,腺伴随病毒载体介导目的基因是治疗ROP视网膜新生血管较为理想的载体。 "     

Immune responses to retinal gene therapy using adeno-associated viral vectors – Implications for treatment success and safety

Recombinant adeno-associated virus (AAV) is the leading vector for gene therapy in the retina. As non-pathogenic, non-integrating, replication deficient vector, the recombinant virus efficiently transduces all key retinal cell populations. Successful testing of AAV vectors in clinical trials of inherited retinal diseases led to the recent approval of voretigene neparvovec (Luxturna) for the treatment of RPE65 mutation-associated retinal dystrophies. However, studies applying AAV-mediated retinal gene therapy independently reported intraocular inflammation and/or loss of efficacy after initial functional improvements. Both observations might be explained by targeted removal of transduced cells via anti-viral defence mechanisms. AAV has been shown to activate innate pattern recognition receptors (PRRs) such as toll-like receptor (TLR)-2 and TLR-9 resulting in the release of inflammatory cytokines and type I interferons. The vector can also induce capsid-specific and transgene-specific T cell responses and neutralizing anti-AAV antibodies which both limit the therapeutic effect. However, the target organ of retinal gene therapy, the eye, is known as an immune-privileged site. It is characterized by suppression of inflammation and promotion of immune tolerance which might prevent AAV-induced immune responses. This review evaluates AAV-related immune responses, toxicity and inflammation in studies of retinal gene therapy, identifies influencing variables of these responses and discusses potential strategies to modulate immune reactions to AAV vectors to increase the safety and efficacy of ocular gene therapy.     

Adeno-associated viruses containing bFGF or BDNF are neuroprotective against excitotoxicity

PURPOSE: Brain-derived neurotrophic factor (BDNF) and basic fibroblast growth factor (bFGF) hold much promise for the protection of retinal ganglion cells against excitotoxic cell death. We tested the possibility of delivering these growth factors to retinal ganglion cells via an adeno-associated viral (AAV) vector and tested their efficacy in two models of excitotoxicity. METHODS: Rat retinas were infected with AAV vectors encoding bFGF or BDNF. A control vector containing green fluorescent protein (GFP) was injected in the contralateral eye. Eyes were subjected to either an intravitreal injection of N-methyl-D-aspartate (NMDA) or optic nerve crush, and ganglion cell survival was evaluated. RESULTS: AAV.CMV.bFGF and AAV.CBA.BDNF were neuroprotective against NMDA injection 1 month post-treatment. Additionally, AAV.CMV.bFGF was protective against optic nerve crush. CONCLUSION: AAV-mediated delivery of bFGF and BDNF can promote retinal cell survival following excitotoxic insult.     

Long-term safety of GDNF gene delivery in the retina

PURPOSE: To examine retinal function after the long-term, gene therapy-delivered expression of exogenous glial cell line-derived neurotrophic factor (GDNF). METHODS: Forty Sprague-Dawley rats each received an intravitreal injection of recombinant adeno-associated virus expressing GDNF (rAAV-GDNF) in their right eye. The left eye was untreated. One year after viral transduction in ocular tissues, retinal morphology and function were compared between rAAV-GDNF-injected and normal na?ve eyes. Synthesis and accumulation of GDNF within the retina were immunohistologically confirmed using enzyme-linked immunosorbent assay. Morphological analyses included light microscope examination of retinal sections and the counting of retinal ganglion cells. Inflammation by infiltration of leukocytes in retina was assessed immunohistochemically. Retinal function was assessed using electroretinography. RESULTS: GDNF expression was confirmed. There was no obvious abnormality in retinal section or increased infiltration by leukocytes after retinal transduction of rAAV-GDNF for 1 year. Counts of retinal ganglion cells were not decreased in rAAV-GDNF-injected eyes. There were no statistical differences in amplitude as well as latency of the electroretinogram-determined a- and b-waves between transduced and untreated eyes. CONCLUSIONS: Long-term expression of GDNF within the eyes can be achieved by intravitreal injection of rAAV vectors in the absence of morphological or functional deficits in the retina.     

Lentiviral transduction of green fluorescent protein in retinal epithelium: evidence of rejection

This paper demonstrates lentiviral transduction of the humanized form of the Aequoria victoria gene for green fluorescent protein (GFP) into human fetal retinal pigment epithelium (RPE) in vitro and rabbit RPE in vivo.In vitro GFP expression of cultured human fetal RPE begins within two to three days after 12-16 h of maintained exposure to the virus at titers of 10(8)-10(9) infectious units (IU)/ml. Both stationary and dividing cells are transduced using a lenti viral vector with a cytomegalovirus (CMV) promoter. Expression remains stable for at least three to four months without evidence of toxicity and continues through cell division.In vivo expression is followed non-invasively in rabbit eye using a scanning laser ophthalmoscope (SLO), which can detect single fluorescing retinal cells. In vivo expression begins within a few days after a viral solution is introduced into the subretinal space. A solution of 10(9) IU/ml produces fluorescence within three to four days. Less concentrated solutions lead to slower and less expression. No expression is detectable at concentrations of 10(6) IU/ml. Within one to two weeks after introduction of the viral solution, there is evidence of rejection seen by SLO as a loss of GFP fluorescence and disruption of the RPE. Histology shows damage to the RPE layer and monocytic cell infiltrates in the choroid and subretinal space within the area receiving the viral solution. Strong GFP expression leads to rejection within two weeks. With less expression, rejection is delayed and in some cases undetectable for at least six months. If the GFP gene is not included in the viral vector or if the viral concentration is insufficient to produce detectable GFP expression, rejection is not seen. Using a rhodopsin promoter or injecting the virus intra rather than subretinally produces weak expression and no rejection. Lentivirus can induce expression of a foreign gene in the RPE. Viral induced transduction and GFP expression have no effect on the viability of the RPE in vitro. Continued expression of GFP after cell division implies chromosomal integration of the gene. In vivo expression of GFP in RPE encounters rejection. Rejection may not occur with low GFP expression. The latter occurs with low viral titers, a rhodopsin promoter or intra-retinal injection of viral solution. The results are relevant to gene therapy in retina when gene transduction leads to the expression of foreign proteins.