CN114848850A - Rs1基因在制备xlrs治疗剂中的应用及治疗剂 - Google Patents
Rs1基因在制备xlrs治疗剂中的应用及治疗剂 Download PDFInfo
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Abstract
本发明涉及RS1基因在制备X连锁视网膜劈裂症治疗剂中的应用;还涉及一种X连锁视网膜劈裂症的治疗剂,包含RS1基因表达载体,RS1基因表达载体中含有RS1基因表达框。RS1基因表达框中RS1基因序列为经过密码子优化的序列,控制RS1基因表达的启动子为CMV启动子,RS1基因表达框中还包括非编码调节序列。本发明通过构建高效表达的RS1基因表达载体获得了可用于有效治疗X连锁视网膜劈裂症的治疗剂,该治疗剂通过注射的方式施用至玻璃体腔中后,可显著提高RS1蛋白的表达,从而达到治疗X连锁视网膜劈裂症的目的,有希望成为X连锁视网膜劈裂症治疗的候选药物。
Description
技术领域
本发明涉及眼科疾病治疗和分子药物领域,具体涉及RS1基因在制备X 连锁视网膜劈裂症治疗剂中的应用。
背景技术
X连锁视网膜劈裂症(X-linked juvenile retinoschisis,XLRS)是一种RS1基因突变引起的罕见视网膜变性疾病,发病率约为1:25,000~ 1:5,000。该病呈X染色体隐性遗传,男性发病多见,女性常为无临床症状携带者(George N D,et al.X linkedretinoschisis.Br J Ophthalmol, 1995,79(7):697-702.George N D,et al.Clinicalfeatures in affected males with X-linked retinoschisis.Arch Ophthalmol,1996,114(3): 274-80.)。视网膜劈裂常发生于视网膜神经纤维层与神经节细胞层之间,可导致患者严重的视力下降、黄斑中心凹劈裂以及视网膜电图中b波相对于a 波不成比例的下降(Molday R S,et al.X-linked juvenile retinoschisis: clinical diagnosis,geneticanalysis,and molecular mechanisms.Prog Retin Eye Res,2012,31(3):195-212.)。目前临床上多以观察和并发症的治疗为主,尚无有效的治疗方法。
随着越来越多的治疗中将腺相关病毒(AAV)作为递送载体,基因治疗作为一种根本性治疗方法为XLRS的治疗提供了新的可能性。但是,就目前而言,尚未构建和筛选出足够有效的递送载体将正常RS1基因递送至机体内,用于基因治疗XLRS。
发明内容
为解决以上问题,本发明提供了RS1基因在制备X连锁视网膜劈裂症治疗剂中的应用。
本发明还提供了一种X连锁视网膜劈裂症的治疗剂,包含RS1基因表达载体,所述RS1基因表达载体中含有RS1基因表达框。
在一个具体实施方案中,所述RS1基因表达框中RS1基因序列为经过密码子优化的序列:如SEQ ID NO:6所示。密码子优化后的RS1基因表达框在体外表达实验中,其mRNA水平是原RS1基因表达框5倍左右,蛋白水平是7 倍左右。
在一个具体实施方案中,控制所述RS1基因表达的启动子为CMV启动子。
在一个具体实施方案中,所述RS1基因表达框中还包括非编码调节序列,所述非编码调节序列包括Intron序列、Kozak序列、5’UTR序列和WPRE序列和HGHpA序列中的一种或多种组合。
在一个优选实施方案中,所述Intron序列如SEQ ID NO:4所示,所述 Kozak序列如SEQ ID NO:5所示,所述WPRE序列如SEQ ID NO:7所示,所述 HGHpA序列如SEQ ID NO:3所示,所述5’UTR序列选自SEQ ID NO:8和9 或其由SEQ ID NO:8和9串接而成。在串接的实施方案中,5’UTR序列如 SEQ ID NO:10。在表达载体构建过程中,我们通过密码子优化和非编码调节序列的设计,共同推高了RS1基因表达载体的表达效率,使其的mRNA水平和蛋白水平提高至现有表达载体的12-16倍。
在一个优选实施方案中,所述RS1基因表达框为 CMV-Intron-5’UTR-Kozak-hRS1-WPRE-HGHpA。
在一个具体实施方案中,所述表达载体为腺相关病毒表达载体,包装在腺相关病毒颗粒中。
优选地,所述腺相关病毒为AAV2/8。
本发明通过构建高效表达的RS1基因表达载体获得了可用于有效治疗X 连锁视网膜劈裂症的治疗剂,该治疗剂通过注射的方式施用至玻璃体腔中后,可显著提高RS1蛋白的表达,从而达到治疗X连锁视网膜劈裂症的目的,有希望成为X连锁视网膜劈裂症治疗的候选药物。
附图说明
图1为表达载体1的质粒谱图。
图2为表达载体2的质粒谱图。
图3为表达载体3的质粒谱图。
图4为表达载体4的质粒谱图。
图5为表达载体5的质粒谱图。
图6为HEK293T细胞被包装了表达载体1-5的AAV病毒颗粒分别感染后, RS1基因的mRNA水平相对表达量和蛋白水平相对表达量的统计图。
图7为各组小鼠的视网膜HE染色图和视网膜厚度统计图。
图8为各组小鼠在暗视和明视条件下的视动反应统计图(A)和视敏度统计图(B),在B图中,每一簇柱子从左至右依次是对照组、治疗模型组和非治疗模型组。
图9为各组小鼠的ERG(视网膜电图)波形图(A)及波幅统计图(B)。
具体实施方式
以下结合附图对发明的原理和特征进行描述,所举实例只用于解释发明,并非用于限定发明的范围。
1、AAV-RS1重组表达载体的构建
我们在研究中发现,使用不同的RS1表达载体对视网膜劈裂症可产生不同的治疗效果。为了找到最优的表达载体,我们以pAAV-MCS-ITR为骨架设计了三代AAV重组表达载体,第一代为表达载体1,第二代为表达载体2,第三代为表达载体3-5。
表达载体1和2的结构分别如图1和2所示,为 rAAV2/8-CMV-hRS1-HGHpA,其中CMV是指CMV启动子,序列如SEQ ID NO:1 所示,HGHpA为人生长激素多聚腺苷酸,序列如SEQ IDNO:3所示;区别在于,使用了不同的RS1编码序列,其中表达载体1使用的hRS1wt为RS1野生型序列,序列如SEQ ID NO:2所示,表达载体2使用的hRS1sgOPT为密码子优化的RS1基因,序列如SEQ ID NO:6所示。
表达载体3-5的结构如图3-5所示,为rAAV2/8-CMV-Intron-5’UTR- Kozak-hRS1sgOPT-WPRE-HGHpA,其中,CMV是指CMV启动子,内含子(intron) 为真核生物细胞DNA中的间插序列,该序列被转录在前体RNA中,经过剪接被去除,最终不存在于成熟RNA分子中。我们采用了一种人工合成的嵌合内含子,能够增强mRNA的加工过程并提高下游基因编码框的表达水平,如SEQ ID NO:4所示。在真核表达系统中,Kozak序列通常可以提高转录和翻译的效率,如SEQ ID NO:5所示;hRS1sgOPT为密码子优化的RS1基因;土拨鼠肝炎病毒转录后调节元件(WPRE)是一种DNA序列,当转录时会产生增强表达的三级结构,能够增加病毒载体递送的外源基因的表达水平。WPRE的序列如SEQ ID NO:7所示;HGHpA为人生长激素(hGH)多聚腺苷酸(PolyA),在 mRNA核外转运和翻译过程中起维持mRNA稳定、避免其被降解的重要作用。
有研究表明合理设计5’非翻译区(5’UTR)可以提高外源蛋白的表达水平。表达载体3-5的区别在于5’UTR不同,实验中我们设计了多个5’UTR 序列,结果发现5’UTR-2(SEQID NO:8)和5’UTR-3(SEQ ID NO:9)具有较好的效果,表达载体3的5’UTR为5’UTR-2,表达载体4的5’UTR 为5’UTR-3,表达载体5的5’UTR为串接的5’UTR-2和5’UTR-3,两条序列中间用linker连接,即5’UTR-2-3(SEQ ID NO:10)。
2、AAV病毒的制备与纯化
我们采用PEI转染试剂将3种质粒,即:AAV8的Rep及Cap蛋白表达质粒(pAAV-RC2/8),辅助质粒(pAd-helper)和携带有外源基因的AAV核心表达质粒,共转染HEK293细胞包装制备AAV病毒。转染96h后,收获细胞与培养上清。在切向过滤(TFF)浓缩病毒粗纯液后,使用碘克沙醇密度梯度超速离心的方法对AAV病毒进行进一步纯化,然后使用Amicon Ultra100K 的透析管离心进行脱盐和浓缩。利用荧光定量PCR(Q-PCR)法测定纯化病毒的滴度,通过SDS-PAGE染色检测病毒的纯度,以及鲎试剂检测内毒素含量。最终,我们将AAV病毒的滴度调整至2×1012vg/mL。纯化的AAV病毒分装后存储于-80℃冰箱。
3、AAV病毒感染HEK293T细胞体外表达效率的比较
1)HEK293T细胞的感染:取6孔板分为5个实验组,分别接种5×105的HEK293T细胞在每个孔内,接种1天后,细胞计数1×106左右,分别感染 50μl(1×1010vg)的AAV病毒,表达载体1病毒颗粒(A组)、表达载体 2病毒颗粒(B组)、表达载体3病毒颗粒(C组)、表达载体4病毒颗粒(D 组)和表达载体5病毒颗粒(E组)。感染48小时后,分别提取被感染细胞的mRNA和蛋白进行检测分析。
2)Q-PCR检测RS1基因在mRNA水平相对表达量:首先利用Trizol试剂盒提取细胞总RNA并反转录合成cDNA模板。然后,利用荧光定量PCR进行检测。Q-PCR反应体系:SYBR GreenⅡ10μl;目的片段引物各0.3μl; ROX 0.4μl;无菌水加至20μl。Q-PCR反应程序为:预变性95℃5min;循环40次:95℃15s,60℃1min。采用2-ΔΔCt相对定量的方法分析RS1 基因表达量的差异。
3)Western blot检测RS1基因在蛋白水平相对表达量:分离不同组别的HEK293T细胞,去除培养液后用PBS洗一遍,加入裂解液充分裂解后,12000g离心5分钟,取上清。利用BCA法测定蛋白浓度,然后按总蛋白 30μg计算各组的上样体积,进行SDS-PAGE凝胶电泳和Western blot检测,用Image J软件进行灰度定量分析。
结果如图6所示,无论是mRNA水平还是蛋白水平,目的蛋白的相对表达量,B、C、D、E组均比A组均有显著提高,其中B组提高了约7倍以上, C、D、E组提高了约12倍以上。由此可见,我们通过密码子优化大幅提高了 RS1基因的表达量,并通过设计非编码的调控序列进一步推升了RS1基因的表达量。经过优化后,第二代和三代表达载体具有高得多的体外感染和表达效率,尤其是E组(两个5’UTR串接之后的表达载体5)的表达量是A组(表达载体1)的16倍以上。因此,后面的动物实验选用表达载体5进行。
4、XLRS模型小鼠RS1h-KO构建
我们通过CRISPR/Cas9技术敲除小鼠RS1同源基因RS1h的第一外显子和1.6×103bp大小的部分第一内含子获得了XLRS模型小鼠RS1h-KO。简要过程如下:首先制备针对靶基因RS1h的Cas9 mRNA和sgRNA,再通过显微注射技术将其直接注射到0.5天的C57BL/6J小鼠受精卵中。最后将受精卵移植到假孕母体的输卵管内,获得F0代小鼠。通过PCR基因型鉴定和测序验证筛选出满足要求的F0代小鼠,随后将其同C57BL/6J小鼠配繁建系,获得稳定的F1代阳性鼠。子代小鼠剪鼠尾经PCR技术进行鉴定。实验所用野生型C57BL/6J小鼠均购于北京维通利华实验动物技术有限公司。所有小鼠均饲养于武汉大学医学研究院动物实验中心SPF级屏障环境(22±2℃,60±10%相对湿度,12h/12h明暗循环)中饲养及繁殖,自由进食进水。所有动物实验和程序均经武汉大学医学部医学研究院动物伦理委员会批准,并严格遵循《实验动物管理和使用指南》。
5、RS1表达载体病毒颗粒对RS1h-KO小鼠视网膜结构和视功能的恢复
将表达载体5制备的RS1表达载体病毒颗粒通过眼内局部给药,即视网膜下腔或玻璃体腔注射给药。本发明中将重组病毒制剂1μl通过玻璃体腔直接注射到P22±3的RS1h-KO小鼠眼内,使小鼠视网膜表达人源RS1蛋白,从而实现视网膜结构和视功能的恢复。具体步骤:小鼠经复方托比卡胺滴眼液充分散瞳后用5%水合氯醛全身麻醉,在解剖显微镜直视下,使用自组装的微量注射器和32G的胰岛素诺和针头在角巩膜缘后方1mm斜向进针至玻璃体腔,注意避开虹膜和晶状体,缓慢推注1μl剂量,并停针2分钟。术后涂左氧氟沙星眼膏,连续三天,每天一次,以减少炎症反应、防止感染。
在治疗后4周,采用HE染色、视动和ERG检测RS1h-KO小鼠视网膜结构和视功能的恢复。
5.1视网膜结构
通过HE染色来检测视网膜结构:深度麻醉后颈椎脱臼法处死小鼠,摘取眼球,PBS漂洗,4%多聚甲醛固定24h。常规梯度乙醇脱水后,60℃二甲苯中浸泡10-15分钟,石蜡包埋。沿平行于视神经的矢状轴切片,厚度为4 μm。HE染色,封片后光学显微镜下观察。应用Image J软件测量距视神经 500μm两侧的内核层、外核层和全视网膜厚度。
结果如图7A所示,经治疗4周后,RS1h-KO小鼠的各层结构排列恢复接近正常,劈裂囊腔几乎完全消失,而对侧眼视网膜结构稍显紊乱,且仍有劈裂囊腔的存在。图7B提示RS1h-KO小鼠治疗眼视网膜内核层(INL)厚度显著下降,接近正常水平。
5.2视动反应
通过以下方法检测视动反应:在黑暗中适应3-4小时(暗适应条件)或在400lux光照刺激下适应10分钟(明适应条件)后,小鼠被放置于载鼠台(直径8.5cm,距底部镜面高度17.5cm,位于镜面中心位置)上自由活动。载鼠台上下方是两面镜子(上方镜面中央掏空一直径24cm圆),顶部镜子上放置摄像头进行记录。载鼠台四周由四块液晶显示器组成,可显示不同频率的移动光栅(光栅的空间频率从0.1增加到0.6cyc/deg:0.1、0.15、 0.2、0.25、0.3、0.35、0.4、0.45、0.5、0.6cyc/deg;每个频率下的光栅分别以顺时针和逆时针方向交替旋转,各方向持续30秒,期间暂停10秒)。小鼠看到周围移动光栅时会作出转动头部的追踪行为。通过分析不同频率移动光栅条件下小鼠的头部转动情况进行视敏度量化。
结果如图8所示,RS1h-KO小鼠治疗眼在暗视和明视条件下的视动反应中的转头次数均大于对侧眼,且部分差异有统计学意义(图8A)。同时,其治疗眼在暗视和明视条件下的视力也均表现出显著提升(图8B)。
5.3ERG反应
通过以下方法检测ERG反应:黑暗环境中过夜(暗视条件)或在系统背景光下持续刺激10分钟后(明视条件),在小鼠双眼表面滴复方托吡卡胺眼药水进行散瞳。随后以0.01mL/g的剂量腹腔注射5%水合氯醛麻醉小鼠,麻醉后外用盐酸羟布普卡因滴眼液进行眼表麻醉。待小鼠进入麻醉状态后连接电极,记录电极垂直轻微接触小鼠角膜中心顶端,参考电极和接地电极分别插入小鼠两耳之间皮下和尾部。所有操作均在暗红光下进行以保证小鼠的暗适应状态。使用RetiMINER 4.0眼电生理视觉系统进行测量,依次增加光强刺激并记录视网膜综合电位反应。a波振幅是从基线到a波波谷的电位差, b波振幅是从a波波谷到b波波峰的电位差。
结果如图9所示,RS1h-KO小鼠治疗眼在ERG检测中所有光强下的a、b 波振幅均高于对侧眼,且在几乎所有中高光强,甚至部分低光强条件下有统计学意义。总体来看,RS1h-KO小鼠经病毒玻璃体腔注射治疗4周后在最高光强条件下的a、b波振幅大致可恢复到正常小鼠的一半水平。
使用表达载体3和4也能取得类似的体内实验结果,使用表达载体2虽然效果比表达载体1好,但是不及第三代表达载体(表达载体3-5)。
尽管上面已经示出和描述了发明的实施例,可以理解的是,上述实施例是示例性的,不能理解为对发明的限制,本领域的普通技术人员在发明的范围内可以对上述实施例进行变化、修改、替换和变型。
序列表
<110> 武汉大学
<120> RS1基因在制备XLRS治疗剂中的应用及治疗剂
<160> 10
<170> SIPOSequenceListing 1.0
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acgacccccg cccattgacg tcaataatga cgtatgttcc catagtaacg ccaataggga 180
ctttccattg acgtcaatgg gtggagtatt tacggtaaac tgcccacttg gcagtacatc 240
aagtgtatca tatgccaagt acgcccccta ttgacgtcaa tgacggtaaa tggcccgcct 300
ggcattatgc ccagtacatg accttatggg actttcctac ttggcagtac atctacgtat 360
tagtcatcgc tattaccatg gtgatgcggt tttggcagta catcaatggg cgtggatagc 420
ggtttgactc acggggattt ccaagtctcc accccattga cgtcaatggg agtttgtttt 480
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gattgccaag gaggacccaa tgctctgtgg tctgcaggtg ccacctcctt ggactgtata 180
ccagaatgcc catatcacaa gcctctgggt ttcgagtcag gggaggtcac accggaccag 240
atcacctgct ctaacccgga gcagtatgtg ggctggtatt cttcgtggac tgcaaacaag 300
gcccggctca acagtcaagg ctttgggtgt gcctggctct ccaagttcca ggacagtagc 360
cagtggttac agatagatct gaaggagatc aaagtgattt cagggatcct cacccagggg 420
cgctgtgaca tcgatgagtg gatgaccaag tacagcgtgc agtacaggac cgatgagcgc 480
ctgaactgga tttactacaa ggaccagact ggaaacaacc gggtcttcta tggcaactcg 540
gaccgcacct ccacggttca gaacctgctg cggcccccca tcatctcccg cttcatccgc 600
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gggtggcatc cctgtgaccc ctccccagtg cctctcctgg ccctggaagt tgccactcca 60
gtgcccacca gccttgtcct aataaaatta agttgcatca ttttgtctga ctaggtgtcc 120
ttctataata ttatggggtg gaggggggtg gtatggagca aggggcaagt tgggaagaca 180
acctgtaggg cctgcggggt ctattgggaa ccaagctgga gtgcagtggc acaatcttgg 240
ctcactgcaa tctccgcctc ctgggttcaa gcgattctcc tgcctcagcc tcccgagttg 300
ttgggattcc aggcatgcat gaccaggctc agctaatttt tgtttttttg gtagagacgg 360
ggtttcacca tattggccag gctggtctcc aactcctaat ctcaggtgat ctacccacct 420
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<210> 4
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atgagcagaa agatcgaggg attcctgctg ctgctgctgt ttggctatga ggccaccctg 60
ggcctgtcta gtaccgagga tgagggagaa gatccttggt atcagaaggc ctgtaaatgt 120
gattgtcagg gcggccctaa tgccctgtgg agcgccggag ctaccagcct ggattgtatt 180
cctgagtgtc cttaccacaa gcctctggga tttgagagcg gagaagtgac ccctgatcag 240
atcacctgta gcaatcctga gcagtatgtg ggctggtact cttcttggac agctaataag 300
gccagactga atagccaggg atttggctgc gcttggctga gcaagtttca ggactctagc 360
cagtggctgc agatcgatct gaaggaaatc aaagtgattt ccggaatctt gacccaggga 420
agatgtgata tcgatgagtg gatgaccaag tacagcgtgc agtatagaac agatgagaga 480
ctgaattgga tctattataa ggaccagaca ggcaataata gagtgtttta cggcaattct 540
gatagaacaa gcaccgtgca gaatctgctg agaccaccta ttatttctag gtttatcaga 600
ctgattcctc tgggctggca cgtgagaatc gccatcagaa tggagctgct ggagtgcgtg 660
agcaagtgtg cctga 675
<210> 7
<211> 600
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 7
gcttatcgat aatcaacctc tggattacaa aatttgtgaa agattgactg gtattcttaa 60
ctatgttgct ccttttacgc tatgtggata cgctgcttta atgcctttgt atcatgctat 120
tgcttcccgt atggctttca ttttctcctc cttgtataaa tcctggttgc tgtctcttta 180
tgaggagttg tggcccgttg tcaggcaacg tggcgtggtg tgcactgtgt ttgctgacgc 240
aacccccact ggttggggca ttgccaccac ctgtcagctc ctttccggga ctttcgcttt 300
ccccctccct attgccacgg cggaactcat cgccgcctgc cttgcccgct gctggacagg 360
ggctcggctg ttgggcactg acaattccgt ggtgttgtcg gggaaatcat cgtcctttcc 420
ttggctgctc gcctatgttg ccacctggat tctgcgcggg acgtccttct gctacgtccc 480
ttcggccctc aatccagcgg accttccttc ccgcggcctg ctgccggctc tgcggcctct 540
tccgcgtctt cgccttcgcc ctcagacgag tcggatctcc ctttgggccg cctccccgca 600
<210> 8
<211> 100
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 8
cactcgcgct gccatcactc ttccggcgtc ttcgccgcca tcctcgccgc gactcgcttc 60
tttcggttct accaggtaga gtccggcgcc atcctccacc 100
<210> 9
<211> 100
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 9
cttgtctcgc tccggggaac gctccgaaac tcccggccgc cgccaccggc gtctgttctg 60
ttacacaagg caagaaaagc cgctgccgca ctccgagtgt 100
<210> 10
<211> 206
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 10
cactcgcgct gccatcactc ttccggcgtc ttcgccgcca tcctcgccgc gactcgcttc 60
tttcggttct accaggtaga gtccggcgcc atcctccacc caacaacttg tctcgctccg 120
gggaacgctc cgaaactccc ggccgccgcc accggcgtct gttctgttac acaaggcaag 180
aaaagccgct gccgcactcc gagtgt 206
Claims (10)
1.RS1基因在制备X连锁视网膜劈裂症治疗剂中的应用。
2.一种X连锁视网膜劈裂症的治疗剂,其特征在于,包含RS1基因表达载体,所述RS1基因表达载体中含有RS1基因表达框。
3.根据权利要求2所述的治疗剂,其特征在于,所述RS1基因表达框中RS1基因序列为经过密码子优化的序列,如SEQ ID NO:6所示。
4.根据权利要求2所述的治疗剂,其特征在于,控制所述RS1基因表达的启动子为CMV启动子。
5.根据权利要求3所述的治疗剂,其特征在于,所述RS1基因表达框中还包括非编码调节序列,所述非编码调节序列包括Intron序列、Kozak序列、5’UTR序列和WPRE序列和HGHpA序列中的一种或多种组合。
6.根据权利要求5所述的治疗剂,其特征在于,所述RS1基因表达框为CMV-Intron-5’UTR-Kozak-hRS1-WPRE-HGHpA。
7.根据权利要求5所述的治疗剂,其特征在于,所述Intron序列如SEQ ID NO:4所示,所述Kozak序列如SEQ ID NO:5所示,所述WPRE序列如SEQ ID NO:7所示,所述HGHpA序列如SEQID NO:3所示,所述5’UTR序列选自SEQ ID NO:8和9,或者由SEQ ID NO:8和9串接而成。
8.根据权利要求7所述的治疗剂,其特征在于,所述5’UTR由SEQ ID NO:8和9串接而成,序列如SEQ ID NO:10所示。
9.根据权利要求2-8中任一项所述的治疗剂,其特征在于,所述表达载体为腺相关病毒表达载体,包装在腺相关病毒颗粒中。
10.根据权利要求9所述的治疗剂,其特征在于,所述腺相关病毒为AAV2/8。
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Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN115851745A (zh) * | 2022-10-19 | 2023-03-28 | 河南省人民医院 | 调控Esrrb活性的物质在制备用于干预视网膜劈裂症的产品中的应用 |
CN115968834A (zh) * | 2023-01-20 | 2023-04-18 | 北京因诺惟康医药科技有限公司 | 一种rs1点突变小鼠模型的制备方法及其用途 |
WO2023207672A1 (zh) * | 2022-04-28 | 2023-11-02 | 武汉中眸生物科技有限责任公司 | Rs1基因在制备xlrs治疗剂中的应用及治疗剂 |
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CN117683779B (zh) * | 2024-01-26 | 2024-04-30 | 广州嘉检医学检测有限公司 | 视网膜劈裂症相关的基因突变体及其应用 |
Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150259395A1 (en) * | 2014-03-17 | 2015-09-17 | Avalanche Biotechnologies, Inc. | Compositions and methods for enhanced gene expression in cone cells |
WO2019241684A1 (en) * | 2018-06-15 | 2019-12-19 | Massachusetts Institute Of Technology | Synthetic 5' utr sequences, and high-throughput engineering and screening thereof |
CN111621502A (zh) * | 2019-02-28 | 2020-09-04 | 武汉纽福斯生物科技有限公司 | 视网膜劈裂蛋白的编码序列、其表达载体构建及其应用 |
CN113025657A (zh) * | 2021-03-05 | 2021-06-25 | 中国科学院精密测量科学与技术创新研究院 | 一种可用于双模态成像的腺相关病毒及其应用 |
CN113025618A (zh) * | 2019-12-24 | 2021-06-25 | 上海市第一人民医院 | 一种x连锁遗传性视网膜劈裂症的基因治疗方案与应用 |
CN113322281A (zh) * | 2021-05-12 | 2021-08-31 | 成都金唯科生物科技有限公司 | 一种高效组织特异性表达rs1蛋白的重组腺相关病毒及应用 |
WO2022026632A2 (en) * | 2020-07-29 | 2022-02-03 | University Of Florida Research Foundation, Incorporated | Improved aav-mediated x-linked retinoschisis therapies |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
EP3452101A2 (en) * | 2016-05-04 | 2019-03-13 | CureVac AG | Rna encoding a therapeutic protein |
CN114848850B (zh) * | 2022-04-28 | 2023-12-15 | 武汉中眸生物科技有限责任公司 | Rs1基因在制备xlrs治疗剂中的应用及治疗剂 |
-
2022
- 2022-04-28 CN CN202210460778.5A patent/CN114848850B/zh active Active
-
2023
- 2023-04-18 WO PCT/CN2023/088958 patent/WO2023207672A1/zh unknown
Patent Citations (7)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US20150259395A1 (en) * | 2014-03-17 | 2015-09-17 | Avalanche Biotechnologies, Inc. | Compositions and methods for enhanced gene expression in cone cells |
WO2019241684A1 (en) * | 2018-06-15 | 2019-12-19 | Massachusetts Institute Of Technology | Synthetic 5' utr sequences, and high-throughput engineering and screening thereof |
CN111621502A (zh) * | 2019-02-28 | 2020-09-04 | 武汉纽福斯生物科技有限公司 | 视网膜劈裂蛋白的编码序列、其表达载体构建及其应用 |
CN113025618A (zh) * | 2019-12-24 | 2021-06-25 | 上海市第一人民医院 | 一种x连锁遗传性视网膜劈裂症的基因治疗方案与应用 |
WO2022026632A2 (en) * | 2020-07-29 | 2022-02-03 | University Of Florida Research Foundation, Incorporated | Improved aav-mediated x-linked retinoschisis therapies |
CN113025657A (zh) * | 2021-03-05 | 2021-06-25 | 中国科学院精密测量科学与技术创新研究院 | 一种可用于双模态成像的腺相关病毒及其应用 |
CN113322281A (zh) * | 2021-05-12 | 2021-08-31 | 成都金唯科生物科技有限公司 | 一种高效组织特异性表达rs1蛋白的重组腺相关病毒及应用 |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2023207672A1 (zh) * | 2022-04-28 | 2023-11-02 | 武汉中眸生物科技有限责任公司 | Rs1基因在制备xlrs治疗剂中的应用及治疗剂 |
CN115851745A (zh) * | 2022-10-19 | 2023-03-28 | 河南省人民医院 | 调控Esrrb活性的物质在制备用于干预视网膜劈裂症的产品中的应用 |
CN115851745B (zh) * | 2022-10-19 | 2023-10-27 | 河南省人民医院 | 调控Esrrb活性的物质在制备用于干预视网膜劈裂症的产品中的应用 |
CN115968834A (zh) * | 2023-01-20 | 2023-04-18 | 北京因诺惟康医药科技有限公司 | 一种rs1点突变小鼠模型的制备方法及其用途 |
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