李斌医生的科普号

Efficacy and Safety of rAAV2-ND4Treatment for Leber’s HereditaryOptic NeuropathyXing Wan1,* , Han Pei1,* , Min-jian Zhao2,* , Shuo Yang1,* , Wei-kun Hu1 , Heng He1 , Si-qi Ma1 ,Ge Zhang3 , Xiao-yan Dong4 , Chen Chen5 , Dao-wen Wang5 & Bin Li1Leber’s hereditary optic neuropathy (LHON) is a mitochondrially inherited disease leading to blindness.A mitochondrial DNA point mutation at the 11778 nucleotide site of the NADH dehydrogenase subunit4 (ND4) gene is the most common cause. The aim of this study was to evaluate the efficacy and safety ofa recombinant adeno-associated virus 2 (AAV2) carrying ND4 (rAAV2-ND4) in LHON patients carryingthe G11778A mutation. Nine patients were administered rAAV2-ND4 by intravitreal injection to one eyeand then followed for 9 months. Ophthalmologic examinations of visual acuity, visual field, and opticalcoherence tomography were performed. Physical examinations included routine blood and urine. Thevisual acuity of the injected eyes of six patients improved by at least 0.3 log MAR after 9 months offollow-up. In these six patients, the visual field was enlarged but the retinal nerve fibre layer remainedrelatively stable. No other outcome measure was significantly changed. None of the nine patientshad local or systemic adverse events related to the vector during the 9-month follow-up period. Thesefindings support the feasible use of gene therapy for LHON.

1.视网膜色素变性这个病是全球性难题，全世界拿这个病没有太好的方法；2.网上查询勿轻信，病急不可乱投医；3.不要做任何的眼部手术，激光等（白内障病人另说）4.叶黄素对病人没有太好的效果，并且抽烟的人和经常抽二手烟的人吃叶黄素会让得肺癌的几率变大；

欧洲一个关于艾地苯醌治疗Leber遗传性视神经病变的多中心研究已经结束，结果发表在英国brain杂志：Arandomized placebo-controlled trial of idebenone in Leber's hereditary opticneuropathy. Brain. 2011，134:2677-86.结果显示：使用艾地苯醌900mg/天，24周，治疗组的患者与使用安慰剂的患者比较，最佳矫正视力并未有显著性提高，统计学分析两组差异没有显著性，简言之，艾地苯醌不能够提高患者视力。相关学者进一步发表相关评论，说明目前没有找到有效的方法治疗Leber遗传性视神经病变。SCIENTIFICCOMMENTARIESTreatment ofLeber hereditary optic neuropathy，Brain 2011: 134;2447–2455。基因治疗将是Leber遗传性视神经病变的治疗方向。

目的 构建一种新的人ND4基因腺相关病毒，可用于研究Leber遗传性视神经病变的基因治疗。 方法 1.人工合成一种新的人正常ND4基因,构建rAAV2/2-ND4重组腺相关病毒,进行纯化与浓缩后,用特异扩增ND4基因的引物进行PCR鉴定,用地高辛标记的H1探针点杂交方法检测病毒液中rAAV2/2-ND4的物理滴度；2.利用免疫荧光检测ND4是否可以在线粒体中表达。.结果 构建的rAAV2/2-ND4重组腺相关病毒能够特异地扩增出ND4目的基因条带,基因组测定物理滴度为：ND4基因为4.4×1010 vg/ml，证明该重组腺病毒携带目的基因；用免疫荧光检测发现新的ND4在线粒体可以表达。 结论 本实验成功的构建了一种新的人ND4基因腺相关病毒,可用于以后Leber遗传性视神经病变的治疗研究。

高晶，石慧，裴晗，万幸，胡维琨，李涛，杜皓，李斌（通讯作者）目的 玻璃体腔注射重组ND4基因的腺相关病毒血清型2(Adenovirus associated virus 2 -NADH dehydrogenase subunit 4,AAV2-ND4)是Leber遗传性视神经病变(Leber hereditary optic neuropathy,LHON)的基因治疗方法,观察基因治疗后不同免疫抑制方案组全身免疫抑制效果及ND4基因表达情况.方法 各实验组(A~D组)均单次玻璃体腔注射4 μL AAV2-ND4,注射后灌胃予以口服药物28 d,A组不予以免疫抑制治疗,B组口服环孢霉素A[起始剂量10 mg/(kg·d),14 d后减半],C组口服强的松[起始剂量5 mg/(kg·d),14 d后减半],D组玻璃体腔同时注射曲安奈德,并口服强的松(剂量同C组),对照组(E组)单次玻璃体腔注射4 μL磷酸缓冲盐溶液.分别于注射AAV2-ND4后3、7、28、56、84 d后检测血清中AAV2和ND4的抗体含量,同时于第84 d观察鼠视网膜上ND4的表达.结果 A~D各组抗ND4基因抗体浓度在第56天时均明显增加,以A、C组为显著;第84天时,除D组外各组抗体浓度均出现不同程度下降.A~D各组抗AAV2抗体浓度在第28天时明显增加,以A、C组为显著;第56天时除B组外各组抗体浓度均出现下降.实验各组的视网膜神经节细胞层在注射第84天仍有明显ND4表达.结论 免疫反应主要集中在玻璃体腔注射AAV2-ND4后56 d内;各个方案的免疫抑制效果均不理想;不同免疫抑制治疗方案对ND4的表达效率无明显影响.

【摘要】：目的构建人ND4基因腺相关病毒,为Leber遗传性视神经病变(leber hereditary optic neuropathy,LHON)的基因治疗做准备。方法人工合成人的正常ND4基因,构建rAAV2/2-ND4重组腺相关病毒,进行纯化与浓缩后,用特异扩增ND4基因的引物进行PCR鉴定,用地高辛标记的H1探针点杂交方法检测病毒液中rAAV2/2-ND4的物理滴度。结果构建的rAAV2/2-ND4重组腺相关病毒能够特异地扩增出ND4目的基因条带,基因组测定物理滴度为400×109vg·mL-1,证明该重组腺病毒携带目的基因。结论本实验成功地构建了人ND4基因腺相关病毒,作为一种先进的载体系统,为今后Leber遗传性视神经病变患者的基因治疗奠定了基础。《眼科新进展》 2012年03期

文章发表在Clinical and Experimental Ophthalmology 2012AbstractPurpose: To study intravitreal injection dose and safety of recombinant adeno-associated virus (AAV)-mediated gene delivery of human NADH dehydrogenase subunit 4 (ND4) in rabbit eyes. Methods: An open reading frame for human ND4 was fused to the mitochondrial targeting sequence and packed into an AAV capsid. AAV-ND4 and AAV-green fluorescent protein (GFP), 0.1 mL AAV-ND4, 0.1 mL AAV-GFP, or 0.1 mL vehicle was delivered to rabbit eyes via intravitreal injection. The safety of recombinant AAV-mediated gene delivery of human ND4 in rabbit eyes was assessed by slit lamp microscope and direct ophthalmoscope, measurements of intraocular pressure and flash visual evoked potential, and optical coherence tomography (OCT). The mRNA and protein expressions of human ND4 in the retina of rabbits were determined with real-time PCR, immunofluorescence, and Western blot. Results: No complications occurred in any of the three treatment groups after the intravitreal injection. At one-month post-injection, no significant difference in the mean thickness of RNFL was found among the three groups: ,Results of the visual evoked potential test showed that there was no difference in the latency of the visual P100 wave among the three groups, Real-time PCR, immunofluorescence, and Western blot analyses verified the expressions of ND4 and GFP in the RNFL.Conclusions: Intravitreal injection of AAV-ND4 expression vectors was effectively and safely performed in our study. The data on the dose and method of intravitreal injection from our study will provide a valuable reference for clinical intravitreal injection of AAV-ND4 for the treatment of Leber’s hereditary optic neuropathy.