CN115379834A - 用于nmnat1相关视网膜变性的基因疗法 - Google Patents
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- CN115379834A CN115379834A CN202180027825.0A CN202180027825A CN115379834A CN 115379834 A CN115379834 A CN 115379834A CN 202180027825 A CN202180027825 A CN 202180027825A CN 115379834 A CN115379834 A CN 115379834A
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Abstract
用于视网膜变性的基因疗法的方法和组合物,该视网膜变性与烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)的突变相关。
Description
优先权要求
本申请要求于2020年3月11日提交的美国临时专利申请序列号62/988,260的权益。前述案的全部内容在此通过引用并入。
联邦资助的研究或开发
本发明是在美国国家卫生研究院授予的拨款号EY012910下由政府支持进行的。政府在本发明中拥有某些权利。
技术领域
本发明涉及用于视网膜变性的基因疗法的方法和组合物,该视网膜变性与烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)的突变相关。
背景技术
NMNAT1相关视网膜变性是一种早发型隐性疾病,在生命的第一个或第二个十年期间该疾病会导致严重的视力丧失1-4。5受影响基因烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)编码一种泛表达酶,该酶对于在细胞核中再生NAD+是至关重要的。1,6核NAD+池对许多细胞过程很重要,这些过程包括与DNA修复、基因表达、细胞信号传导和细胞衰老相关的过程。7-10NMNAT1的至少三十四个突变与视网膜变性相关,10,11这些突变各自都被认为会在不同程度上降低酶活性。4由于基于在Nmnat1敲除小鼠中的研究,tabttwo完全无功能等位基因的遗传被认为是胚胎致死的,因此核NAD+的严重但不完全丧失可能会导致疾病。12虽然其他两种NMNAT同种型,NMNAT2和NMNAT3,分别在胞质溶胶和线粒体中具有相同的NAD+合酶功能,8,13但显而易见的是二者都不能补偿NMNAT1的丧失,12至少在视网膜中的丧失。这种疾病的孤立性质可以部分地通过如下的最新发现来解释:Nmnat1V9M/V9M小鼠的神经视网膜(用于本研究的相同模型)降低了NAD+水平(并伴随有前体水平增加),而其他组织(包括脑组织)中的水平保持不变(Greenwald等人,RD2018摘要)。然而,视网膜具有这种独特易损性的根本原因仍不清楚。
发明内容
本文提供了用于治疗人受试者中由烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)基因中的突变引起的视网膜变性的方法。一般而言,该方法包括将治疗有效量的腺相关病毒(AAV)载体递送至受试者的眼,该载体包含编码人NMNAT1的序列(例如,与SEQ ID NO:3至少80%相同的序列),该序列与在视网膜细胞中,优选在光感受器中驱动表达的启动子可操作地连接。
在一些实施方案中,启动子是CAG、CASI、CMV、RHO或视紫质激酶(GRK1)启动子。
在一些实施方案中,编码NMNAT1的序列与野生型(SEQ ID NO:2)或密码子优化的(SEQ ID NO:1)序列至少80%相同。
在一些实施方案中,载体经由视网膜下注射递送。
此外,本文提供了用于增加人受试者眼中NMNAT1的表达的方法。该方法包括将治疗有效量的腺相关病毒2型(AAV2)载体递送至受试者的眼,该载体包含编码人NMNAT1的序列,该序列与在视网膜中,优选在光感受器细胞中驱动表达的启动子可操作地连接。
在一些实施方案中,启动子是CAG、CASI、CMV、RHO或视紫质激酶(GRK1)启动子。
在一些实施方案中,NMNAT1序列是密码子优化的。
在一些实施方案中,载体经由视网膜下注射递送。
此外,本文提供了一种腺相关病毒2型(AAV2)载体,该载体包含编码人NMNAT1的序列,该序列与在视网膜中,优选在光感受器细胞中驱动表达的启动子可操作地连接。
在一些实施方案中,启动子是CAG、CASI、CMV、RHO或视紫质激酶(GRK1)启动子。
在一些实施方案中,NMNAT1序列是密码子优化的。在一些实施方案中,编码NMNAT1的序列与野生型(SEQ ID NO:2)或密码子优化的(SEQ ID NO:1)序列至少80%相同。
此外,提供了包含本文描述的载体的药物组合物,该药物组合物被配制成用于经由视网膜下注射递送。
本文描述的载体和组合物可以用于例如治疗人受试者眼中由NMNAT1中的突变引起的视网膜变性和/或用于增加人受试者眼中NMNAT1的表达。
除非另有定义,否则本文所用的所有技术和科学术语与本发明所属领域的普通技术人员通常的理解具有相同含义。本文描述了用于本发明的方法和材料;也可以使用本领域已知的其他合适的方法和材料。材料、方法和实例仅是说明性的,并不旨在是限制性的。本文提及的所有公开出版物、专利申请、专利、序列、数据库条目和其他参考文献通过引用以其整体并入本文。在冲突的情况下,应当以本说明书(包括定义)为准。
根据以下详细描述和附图以及权利要求,本发明的其他特征和优点将是明显的。
附图说明
图1A-E.NMNAT1转基因序列和病毒试剂。A)840-核苷酸密码子优化的人NMNAT1cDNA序列(黑色文本,SEQ ID NO:1)具有174个沉默取代(粗体),并且下文示出了各自的野生型核苷酸的身份(WT序列以本文中的SEQ ID NO:2呈现)。B)将由CASI启动子驱动并被包装到SC.AAV2/9中的NMNAT1与EGFP报告构建体共同递送到小鼠中,该报告构建体由相同启动子驱动、随后是WPRE并且被包装在SS.AAV2/9中。C)与图形A相同,不同之处在于将两种构建体都包装在SS.AAV2/9中。D)将由CAG启动子驱动并随后是WPRE的自切割NMNAT1-EGFP融合构建体包装在AAV2/Anc80载体中。E)由CASI启动子驱动并被包装到SS.AAV7m8中的NMNAT1。缩写:bGH,牛生长激素聚腺苷酸化信号;EGFP,增强型绿色荧光蛋白;WPRE,土拨鼠肝炎病毒翻译后调控元件;T2A,明脉扁刺蛾(thesa asigna)病毒2A自切割序列。
图2.体内成像。来自四月龄Nmnat1V9M/V9MM小鼠的被注射(左栏)和未被注射(右栏)视网膜的明视野眼底图像示出了OCT图像中光感受器层测量的平面。白色虚线:下方视网膜(靠近被注射视网膜的注射位点);黄色虚线:上方视网膜(远离被注射眼的注射位点);箭头指示注射位点(顶部行)。EGFP仅在被注射视网膜中可见(中间行)。代表性横截面OCT图像示出,注射2x109gc/μL的SC.AAV2/9试剂的视网膜(左)厚于未被注射的视网膜(右)。
图3A-C.体内成像示出SC.AAV2/9试剂提供了稳定的视网膜结构拯救。A)在注射1x107gc/μL、1x108gc/μL和2x109gc/μL的SC.AAV2/9试剂后,来自九月龄期间的OCT图像的光感受器层厚度测量示出了在更高剂量下的拯救。未被注射Nmnat1V9M/V9MM视网膜的下方区域与同类被注射视网膜的下方和上方区域以及未被注射野生型同窝对照视网膜的下方区域的比较(顶部行)。与被注射同类视网膜的下方和上方区域相比,未被注射野生型视网膜的下方区域的光感受器层厚度测量指示了试剂的毒性最小(底部行)。B)光感受器层厚度测量示出,在两月龄时,在使用1x108gc/μL的SS.AAV2/9试剂的情况下没有检测到拯救。C)光感受器层厚度测量示出,在两月龄时,在使用3x108gc/μL的AAV2/7m8试剂的情况下没有检测到拯救,但光感受器层厚度测量示出,在四月龄期间,在使用5.5x108gc/μL的AAV2/Anc80的情况下存在短暂拯救。误差条表示S.E.M.;*p<0.05,**p<0.1,***p<0.001,****p<0.0001。
图4.高滴度Anc80/Anc80试剂对视网膜有毒。与未被注射野生型小鼠(第一栏)和被注射5.5x108gc/μL剂量的小鼠(第二栏)相比,被注射野生型视网膜(第三栏)和被注射Nmnat1V9M/V9M视网膜(第四栏)的明视野眼底图像(顶部行)示出了视网膜起皱和出血(红色)。通过EGFP表达(中间行)来证实被注射视网膜中的细胞转导。在被注射小鼠中,OCT图像(底部行)示出了视网膜脱离、神经视网膜的破坏以及玻璃体内细胞浸润物(可见为紧邻神经视网膜上方的高反射泪小点(puncta))。小鼠在六周龄时成像。
图5A-B.离体成像示出了用SC.AAV2/9试剂处理的视网膜中细胞水平上的结构拯救。九月龄Nmnat1V9M/V9M和野生型视网膜的图像分别示在左侧和右侧图形组中。A)未被注射(左侧图形)和被注射(中间和右测图形)视网膜的H&E染色。中间和右侧图形示出了与转导主要区域内的注射相关的形态变化性(更多损伤,中间图形;更少损伤,右侧图形)。黑色箭头指示ONL的轻微扇形边。40x mag,比例尺条75μm。B)未被注射视网膜(左栏,两个图形组)既不示出α-NMNAT1抗体反应性(红色)也不示出EGFP表达(绿色),而被注射同类视网膜示出了二者(中间栏,两个图形)。DAPI是合并图像中的复染剂(蓝色)。将示出每个视网膜切片的整体的蒙太奇(montages)用α-NMNAT1抗体标记并且用DAPI复染(左侧和右侧图形组中的右侧图形),以示出跨越视网膜的转基因表达程度。白色箭头指示注射位点。63x放大倍数,比例尺条50μm。
图6.抗人NMNAT1多克隆抗体的验证。在人源ARPE-19细胞(顶部行)中,α-NMNAT1标记(红色,左栏)和DAPI染色的细胞核(蓝色,中间栏)共定位(右栏)。野生型小鼠视网膜示出了与内源性小鼠NMNAT1的最小交叉反应性(中间栏)。在注射AAV后表达人NMNAT1的小鼠视网膜中,在细胞核中检测到免疫反应性(底部行)。放大倍数20x;比例尺条:100μm。
图7A-B.ERG示出了通过用SC.AAV2/9试剂处理保留了视网膜功能。A)与未被处理的同类视网膜相比,来自用2x109gc/μL剂量的SC.AAV2/9处理的Nmnat1V9M/V9M小鼠的视网膜产生显著更大的视杆细胞、混合视杆细胞/视锥细胞和视锥细胞分离ERG,如通过ERG b波所测量的。将来自未被注射Nmnat1V9M/V9M视网膜的测量与被注射同类视网膜以及与野生型小鼠的未被注射和被注射视网膜进行比较(顶部行)。来自六月龄的被处理Nmnat1V9M/V9M小鼠(被处理视网膜,灰色迹线;未被处理视网膜,浅灰色迹线)和年龄匹配的野生型同窝仔(黑色迹线)的代表性ERG波形(底部行)。B)对被注射剂量为1x108gc/μL的SC.AAV2/9的九月龄小鼠的ERG测量示出了比2x109gc/μL剂量更弱的拯救水平(顶部行),并且对被注射剂量为5.5x108gc/μL的AAV2/Anc80的4月龄小鼠的ERG测量指示没有功效(底部行)。误差条表示S.E.M.;*p<0.05,**p<0.1,***p<0.001,****p<0.0001。
图8A-C.成功的疗法需要光感受器中的早期转基因表达。A)注射后14天,在被注射SC.AAV2/9试剂(左栏)的视网膜(尤其是在ONL)中观察到密集的α-NMNAT1免疫反应性(红色),而该信号在年龄匹配的被注射SS.AAV2/9(中间栏)或AA2/Anc80(右栏)试剂的视网膜中是稀疏的。40x放大倍数,比例尺条75μm。B)在p1处用AAV2/Anc80注射来自5.5周龄的野生型小鼠的代表性视网膜,在除视杆细胞光感受器之外的几乎所有细胞类型中都示出了强烈的α-NMNAT1免疫反应性(红色)。ONL中标记细胞的顶部行是视锥细胞(箭头之间),并且DAPI(蓝色)是复染剂。20x放大倍数,比例尺条表示100μm。C).在两周龄时,在对照小鼠中玻璃体内注射AAV2/7m8(3x108gc/μL),在注射后四周,在INL和GCL中产生强烈的NMNAT1表达(红色),但在ONL中没有产生。DAPI是复染剂;20x放大倍数,比例尺条100μm。
具体实施方式
目前,不存在针对NMNAT1相关视网膜变性的治疗。由于患者在生命的最初几年期间会遭受相当大的视力丧失,但预计寿命会正常,因此早期干预有可能使视力保持数十年。由于这种疾病具有隐性遗传模式10,并且由于NMNAT1是一种相对较小的基因,14因此使用腺相关病毒(AAV)介导的基因增补疗法进行的治疗是一种有吸引力的策略。在840bp处,人NMNAT1 cDNA完全在单链AAV(SS.AAV)的约4.7kb最大载货容量15和自身互补AAV(SC.AAV)的约2.2kb最大载货容量16内。这种经由AAV载体向细胞补充突变基因的正常拷贝以维持视网膜细胞活力的方案目前被用作FDA批准的疗法(Luxturna),用于治疗患有RPE65相关视网膜变性的患者。17另外,目前正在进行AAV介导的基因增补疗法的临床试验15以及其他遗传性视网膜变性的大量临床前研究,该基因增补疗法用于无脉络膜症、18全色盲(clinicaltrials.gov,访问日期:11/7/2019)、MERTK视网膜色素变性、19X连锁型视网膜色素变性(recruiting,clinicaltrials.gov,访问日期:11/7/2019)和X连锁型视网膜劈裂症20以及其他病症。21-24
当NMNAT1在2012年首次被报告为疾病基因时,还没有可用于评价潜在的原位疗法的合适动物模型。传统的Nmnat1基因敲除小鼠是不可行的12,并且消除靶向视网膜细胞中的Nmnat1的条件敲除动物不能准确地表示疾病的生理学。本发明人最近报告了NMNAT1相关视网膜疾病小鼠模型的表征,该模型是Nmnat1中p.Val9Met(V9M)突变的纯合子,25其是一种已被发现会导致无关家族成员的视网膜疾病的等位基因。4,10在ENU诱变筛选期间确定了该小鼠系的建立者。纯合子突变后代(Nmnat1V9M/V9M)总是会发展一种早发型孤立性视网膜疾病且不会明显损害寿命、移动性或认知,很像它们所模仿的人类。Nmnat1V9M/V9M小鼠在三周龄时具有完全成熟的视网膜和对光的可靠响应,如通过视网膜电流图(ERG)所检测到的,但一周后,光感受器层示出了变性的迹象,并伴随有功能减少。在大约四月龄时,视网膜严重变性,并且通常无法检测到对光的响应。25考虑到这种小鼠中的突变存在于患者群体中,并且在生命的第一个月期间有机会进行干预,因此该模型适于测试旨在保护视网膜免受NMNAT1相关疾病的疗法。
为了开发一种保持Nmnat1相关视网膜变性患者的视力的疗法,本发明人使用p.V9M-Nmnat1小鼠模型来测试为视网膜细胞提供NMNAT1的正常拷贝可以防止疾病进展的假设。为了实现这一点,将人NMNAT1 cDNA经由若干被独立评价的重组AAV2载体递送至Nmnat1V9M/V9M小鼠的视网膜。功效因病毒制剂和实验条件而变化,并且因此,本发明人旨在了解为何特定变量与成功或失败相关,以及如何推广这些经验以帮助开发其他AAV介导的基因疗法。
从使用AAV介导的基因增补处理的Nmnat1V9M/V9M小鼠中收集的形态学和功能性数据首次证明了任何类型的针对NMNAT1相关视网膜变性的疗法。由于这种小鼠模型的治疗窗口狭窄,因此需要一种自身互补病毒载体,以便可以足够早地表达转基因以拯救脆弱的细胞。自身互补AAV与单链载体的不同之处在于它含有一种反向重复基因组,该基因组折叠以形成双链DNA。42以这种方式,病毒能够绕过转基因表达之前通常需要的第二链DNA合成的限速步骤,31,43以及规避DNA复制后短暂出现的载体基因组不稳定性。44此外,在转基因表达之前,自身互补载体既不需要转运至细胞核,也不需要从衣壳中脱壳。16在这种模型中,单链载体产生了不利的结果,该单链载体包括具有相同AAV血清型/衣壳(AAV2/9)并含有相同基因组货物的单链载体;然而,此类单链载体可以用于包括人在内的其他物种。
NNmnat1V9M/V9M小鼠的治疗窗口受限于早期视杆细胞光感受器发育生物学的限制45,并且受限于后期略小于四周龄时的变性发作。25从逻辑上讲,最早可能的干预将是优选的,这意味着出生后立即治疗。然而,观察到在新生儿注射后视杆细胞未示出强烈的转基因表达,这与其他组的最新报告一致,这些报告描述了P0-P1小鼠中正在发育的视杆细胞光感受器不能被AAV转导。45,46通过将AAV2/7m8的玻璃体内注射递送至两周龄小鼠的实验,清楚地表明了对光感受器中的转基因表达的需要。尽管强烈和广泛覆盖了内层视网膜细胞层,但这种治疗没有提供治疗益处。使用自身互补AAV5,Petit等人报告,如果在P21而不是P10处提供注射,则大约两倍之多的视杆细胞被转导。45
然而,由于SC.AAV2/9需要长于一周的时间来在小鼠视网膜中表达NMNAT1,因此将干预延迟至P21会允许疾病在病毒潜伏期期间无竞争地发展。相反,注射是在约P16处进行的,这为要启动的转基因表达提供了所必需的时间,同时仍然有可能转导很大一部分的视杆细胞群体。在这种情况下,与年龄匹配的野生型同窝仔相比,通过治疗拯救的光感受器的数量足以维持约80%的ONL厚度。应当注意,虽然视网膜结构的保持是即时的,但在注射后3.5个月,ERG才明显拯救功能。
在本方法中,在人患者的治疗窗口期间进行递送。Nmnat1V9M/V9M小鼠中所拯救的视网膜示出,稳定的视锥细胞和视杆细胞光感受器功能保持了数月。虽然来自用SC.AAV2/9治疗的眼的ERG大约是在未被注射野生型视网膜中所测得的值的百分之五十,但这种差异与先前的观察结果一致,即视网膜下注射会抑制ERG响应,这可能是由于手术程序期间持续的机械损伤所致。50这种可能性得到了以下发现的支持:野生型同窝仔的被注射视网膜也倾向于具有b波振幅衰减,但通常较不广泛,以及具有在注射后光感受器层更薄的区域。另外,似乎在病毒潜伏期期间出现的光感受器变性也可能导致较低的信号。替代性假设是,一些细胞被扩充,使得它们过表达NMNAT1,并且这与活力不相容。然而,对NMNAT1/NAD+补充物治疗指数的此种上限效应的原因并不明显。在突变小鼠中,试剂的最高剂量(2x109gc/μL)产生了最佳结果,但由于制造约束,无法测试更高水平的效果。
本方法可以使用普遍活化启动子或细胞类型特异性启动子。p.V9M-Nmnat1小鼠模型的表征指示,光感受器是第一种受到疾病影响的细胞,随后是内层视网膜细胞和RPE,25并且本发明人从此处描述的实验知道,如果要保留视网膜,则必须治疗光感受器。因此,在一些方法中,光感受器特异性启动子用于提供视网膜的拯救。然而,其他细胞类型可能会响应于低核NAD+而以较慢的速率变性,从而导致继发性变性,而仅通过治疗光感受器无法减轻这种变性。在该情况下,则可以使用神经节细胞、双极和Müller神经胶质特异性启动子。
如本文所示,具有驱动NMNAT1表达的CASI的示例性SC.AAV2/9试剂证明了基因增补疗法在患有NMNAT1相关视网膜变性的患者中的成功使用。在一些实施方案中,用自身互补载体快速驱动转基因表达的启动是最佳策略。
这种疗法有可能为世界各地的人们提供数十年的视力,否则他们会在生命早期遭受严重的视力丧失。
载体
本文描述了用于在视网膜中,例如在光感受器中,例如主要或仅在光感受器中体内转染和表达编码如本文所描述的NMNAT1多肽的多核苷酸的靶向表达载体。在一些实施方案中,表达也在内层视网膜细胞或RPE细胞中。可以将此类组件的表达构建体施用于任何有效载体,例如能够有效地将组件基因递送至体内细胞的任何配制剂或组合物。方案包括将基因插入病毒载体,其包括重组逆转录病毒、腺病毒、腺相关病毒、慢病毒和单纯疱疹病毒-1、甲病毒属、牛痘病毒或重组细菌或真核质粒;优选的病毒载体是腺相关病毒2型(AAV2)。病毒载体直接转染细胞;质粒DNA可以以裸露方式或借助于例如以下各项来递送:阳离子脂质体(脂质体转染)或衍生化(例如,抗体缀合)、阳离子树枝状大分子、无机载体(例如,氧化铁磁转染)、类脂质、细胞穿透肽、环糊精聚合物(CDP)、聚赖氨酸缀合物、短杆菌肽S、人工病毒包膜或其他此类细胞内载体,以及在体内进行的基因构建体直接注射或CaPO4沉淀。
用于将核酸体内引入到细胞中的示例性方案是通过使用含有核酸(例如,cDNA)的病毒载体来进行的。用病毒载体感染细胞的优点是大部分靶细胞可以接受核酸。另外,病毒载体内所编码的,例如由该病毒载体中所含有的cDNA编码的分子在已经摄取病毒载体核酸的细胞中有效地表达。
病毒载体可以用作重组基因递送系统,其用于外源性基因体内转移,特别是转移到人体内。这些载体可以有效地将基因递送到细胞中,并且在一些情况下,所转移的核酸稳定地整合到宿主的染色体DNA中。用于产生重组病毒和用于用此类病毒在体外或体内感染细胞的方案可以见于Ausubel,等人,eds.,Gene Therapy Protocols Volume 1:Production and In Vivo Applications of Gene Transfer Vectors,Humana Press,(2008),pp.1-32以及其他标准实验室手册。
可用于递送核酸的优选病毒载体系统是腺相关联病毒(AAV)。腺相关病毒是一种天然存在的缺陷型病毒,其需要另一病毒(如腺病毒或疱疹病毒)作为有效复制和生产生命周期的辅助病毒。(有关综述,参见Muzyczka等人,Curr.Topics in Micro andImmunol.158:97-129(1992);另参见Domenger and Grimm,Human Molecular Genetics,28(R1):R3 R14(2019年10月))。AAV载体有效地转导各种细胞类型,并且可以在体内产生转基因的长期表达。尽管AAV载体基因组可以作为附加体在细胞内持续存在,但已观察到载体整合(参见例如Deyle and Russell,Curr Opin Mol Ther.2009Aug;11(4):442 447;Asokan等人,Mol Ther.2012April;20(4):699 708;Flotte等人,Am.J.Respir.Cell.Mol.Biol.7:349-356(1992);Samulski等人,J.Virol.63:3822-3828(1989);以及McLaughlin等人,J.Virol.62:1963-1973(1989))。AAV载体,特别是AAV2已广泛用于基因增补或替代,并且已经在一系列动物模型以及临床中示出治疗功效;参见,例如,Mingozzi and High,NatureReviews Genetics12,341-355(2011);Deyle and Russell,Curr Opin MolTher.2009Aug;11(4):442 447;Asokan等人,Mol Ther.2012April;20(4):699-708。含有少至300个碱基对的AAV的AAV载体可以包装,并且可以产生重组蛋白表达。外源性DNA的空间限于约4.5kb。例如,AAV1、2、4、5或8载体可以用于将DNA引入到视网膜中,例如引入到光感受器、内层视网膜细胞或RPE细胞(如描述在以下文献中的那些:Maguire等人(2008).Safety and efficacy of gene transfer for Leber's congenital amaurosis.N EnglJ Med 358:2240 2248.Maguire等人(2009).Age-dependent effects of RPE65 genetherapy for Leber'scongenital amaurosis:a phase 1dose-escalation trial.Lancet374:1597 1605;Bainbridge等人(2008).Effect of gene therapy on visual functionin Leber'scongenital amaurosis.N Engl J Med 358:2231 2239;Hauswirth等人(2008).Treatment of leber congenital amaurosis due to RPE65 mutations byocular subretinal injection of adeno-associated virus gene vector:short-termresults of aphase I trial.Hum Gene Ther 19:979 990;Cideciyan等人(2008).Humangene therapy for RPE65 isomerase deficiency activates the retinoid cycle ofvision but with slow rod kinetics.Proc Natl Acad Sci USA 105:1511215117.Cideciyan等人(2009).Vision 1year after gene therapy for Leber'scongenital amaurosis.N Engl J Med 361:725 727;Simonelli等人(2010).Genetherapy for Leber'scongenital amaurosis is safe and effective through1.5years after vector administration.Mol Ther 18:643 650;Acland,等人(2005).Long-term restoration of rod and cone vision by single dose rAAV-mediatedgene transfer to the retina in a canine model of childhood blindness.Mol Ther12:1072 1082;Le Meur等人(2007).Restoration of vision in RPE65-deficientBriard dogs using an AAV serotype 4vector that specifically targets theretinal pigmented epithelium.Gene Ther 14:292 303;Stieger等人(2008).Subretinal delivery of recombinant AAV serotype 8vector in dogs results ingene transfer to neurons in the brain.Mol Ther 16:916 923;以及Vandenberghe等人(2011).Dosage thresholds for AAV2 and AAV8 photoreceptor gene therapy inmonkey.Sci Transl Med 3:88ra54)。在一些实施方案中,AAV载体可以包括WO2015054653中所描述的AAV衣壳多肽(或包括编码该多肽的序列);例如,一种包含AAV衣壳多肽的病毒颗粒,该多肽具有选自由WO 2015054653的SEQ ID NO:1、3、5、7、9、11、13、15和17组成的组的氨基酸序列,以及如本文所描述的编码NMNAT1的序列。在一些实施方案中,AAV衣壳多肽如WO 2015054653的表1所示,在此将该表转载如下:
节点 | 多肽(SEQ ID NO) | 核酸(SEQ ID NO) |
Anc80 | 1 | 2 |
Anc81 | 3 | 4 |
Anc82 | 5 | 6 |
Anc83 | 7 | 8 |
Anc84 | 9 | 10 |
Anc94 | 11 | 12 |
Anc113 | 13 | 14 |
Anc126 | 15 | 16 |
Anc127 | 17 | 18 |
在一些实施方案中,AAV衣壳多肽是Anc80多肽,例如,示出在以下各项中的示例性多肽:SEQ ID NO:19(Anc80L27);SEQ ID NO:20(Anc80L59);SEQ ID NO:21(Anc80L60);SEQID NO:22(Anc80L62);SEQ ID NO:23(Anc80L65);SEQ ID NO:24(Anc80L33);SEQ ID NO:25(Anc80L36);以及SEQ ID NO:26(Anc80L44)。
已经使用AAV载体将多种核酸引入到不同的细胞类型中(参见例如上文引用的参考文献以及以下文献中所引用的那些:Asokan等人,Molecular Therapy(2012);20 4,699-708;以及Hermonat等人,Proc.Natl.Acad.Sci.USA81:6466-6470(1984);Tratschin等人,Mol.Cell.Biol.4:2072-2081(1985);Wondisford等人,Mol.Endocrinol.2:32-39(1988);Tratschin等人,J.Virol.51:611-619(1984);以及Flotte等人,J.Biol.Chem.268:3781-3790(1993)。
在一些实施方案中,使用自身互补AAV,其含有反向重复基因组,该基因组折叠以形成双链DNA。
在一些实施方案中,将编码NMNAT1的基因包埋在其表面带有正电荷的脂质体(例如,脂质体(lipofectin))中,该脂质体可以用针对靶组织的细胞表面抗原的抗体进行标记(Mizuno等人,No Shinkei Geka 20:547-551(1992);PCT公开文本WO91/06309;日本专利申请1047381;以及欧洲专利公开文本EP-A-43075)。
载体还可以包括启动子、增强子(例如,CMV增强子)、其他顺式调控元件和/或衣壳血清型变体。关于启动子,载体可以包括在许多细胞类型(例如,CAG、CMV或CASI)和光感受器细胞(RHO、视紫质激酶(GRK1)和视锥细胞抑制蛋白(CAR))或RPE细胞(例如,RPE特异性蛋白的启动子,如VMD2、RPE65、RLBP1、RGR或TIMP3)中驱动表达的启动子(Esumi等人,JournalBiological Chemistry.2004;279:19064-73;Guziewicz等人,PLoS One.2013;8:e75666;Allocca等人,J Virol.2007;81:11372-80;另参见Domenger and Grimm,Human MolecularGenetics,28(R1):R3 R14(October 2019))。也可以使用合成启动子ProC1和ProD5,参见,例如,Jüttner等人.Nat Neurosci.2019Aug;22(8):1345-1356。其他顺式调控元件可以包括土拨鼠肝炎病毒转录后调控元件(WPRE)或小鼠微小病毒(MVM)内含子(参见Domengerand Grimm,Human Molecular Genetics,28(R1):R3 R14(2019年10月))。
基因疗法构建体的药物制剂可以基本上由可接受稀释剂中的基因递送系统(病毒载体和任何相关试剂,如辅助病毒、蛋白质、脂质等)组成,或者可以包含其中嵌入基因递送载具的缓释基质。替代地,当完整的基因递送系统可以由重组细胞(例如,逆转录病毒载体)完整地产生时,药物制剂可以包含产生基因递送系统的一种或多种细胞。
序列
本方法包括递送编码人NMNAT1的序列。示例性人烟酰胺核苷腺苷酰转移酶1(NMNAT1)序列示出在下表中:
转录物 | 蛋白质 | 同种型编码 | 变体 |
NM_022787.4 | NP_073624.2 | 同种型1 | 变体(1) |
NM_001297778.1 | NP_001284707.1 | 同种型1 | 变体(2) |
NM_001297779.2 | NP_001284708.1 | 同种型2 | 变体(3) |
变体(1)编码较长的同种型(1)。与变体1相比,变体(2)在5'UTR中有所不同。变体1和2编码相同的同种型(1)。与变体1相比,变体(3)缺少外显子并含有替代的3'末端外显子,从而导致不同的3'编码区和3'UTR。经编码的同种型(2)具有不同的C末端,并且比同种型1短。
在一些实施方案中,编码NMNAT1的序列包含SEQ ID NO:2或与SEQ ID NO:2至少80、85、90、95、97、98或99%相同的序列。
人NMNAT1编码序列
在一些实施方案中,编码NMNAT1的序列可以是密码子优化的,使得其可以更有效地被翻译成氨基酸序列。不同生物体的密码子使用表是本领域已知的。示例性密码子优化的编码NMNAT1的序列呈现在图1A/SEQ ID NO:1中。
可用于本方法、载体和组合物的序列包括编码人NMNAT1蛋白或与人NMNAT1蛋白至少80、85、90、95、97、98或99%相同的蛋白质的序列。示例性人NMNAT1蛋白序列呈现在NP_073624.2中,在本文中示出为SEQ ID NO:3。
示例性人NMAT1蛋白序列
在一些实施方案中,可以使用人NMNAT1蛋白同种型2,例如,如在GenBankRef.No.NP_001284708.1处所提供的。
人NMNAT1蛋白可以包括一个或多个突变,例如在多达1、2、3、4、5、10、15或20%的残基处的突变。此类变体应保留野生型蛋白的活性,例如参与细胞核中再生NAD+的能力。在一些实施方案中,突变是保守性取代。此类变化包括用异亮氨酸(I)、缬氨酸(V)和亮氨酸(L)中的任何一种取代这些疏水性氨基酸中的任何其他氨基酸;用天冬氨酸(D)取代谷氨酸(E),反之亦然;用谷氨酰胺(Q)取代天冬酰胺(N),反之亦然;以及用丝氨酸(S)取代苏氨酸(T),反之亦然。其他取代也可以被认为是保守的,这取决于特定氨基酸的环境及其在蛋白质三维结构中的作用。例如,甘氨酸(G)和丙氨酸(A)通常可以互换,丙氨酸(A)和缬氨酸(V)也可以互换。相对疏水的蛋氨酸(M)通常可以与亮氨酸和异亮氨酸互换,并且有时可以与缬氨酸互换。赖氨酸(K)和精氨酸(R)通常可以在其中氨基酸残基的显著特征是其电荷且这两个氨基酸残基的不同pK不显著的位置处互换。在特定环境中,还有一些其他变化可以被认为是“保守的”(参见,例如US20110201052的表III;pages 13-15"Biochemistry"2ndED.Stryer编(Stanford University);Henikoff等人,PNAS1992Vol 89 10915-10919;Lei等人,J Biol Chem 1995May 19;270(20):11882-6)。
为了确定两个氨基酸序列或两个核酸序列的百分比同一性,出于最佳比较目的,对序列进行比对(例如,可以在第一和第二氨基酸或核酸序列中的一个或两个中引入空位以进行最佳比对,并且出于比较目的可以忽略非同源序列)。在优选的实施方案中,出于比较目的,所比对的参考序列的长度是参考序列的长度的至少80%,并且在一些实施方案中是至少90%或100%。然后比较相应氨基酸位置或核苷酸位置处的氨基酸残基或核苷酸。当第一序列中的位置被与第二序列中的相应位置相同的氨基酸残基或核苷酸占据时,则分子在该位置处是相同的(如本文所用,氨基酸或核酸“同一性”与氨基酸或核酸“同源性”等效)。两个序列之间的百分比同一性是被序列共享的相同位置的数目的函数,这考虑到了空位的数目和每个空位的长度,需要引入这些空位以进行两个序列的最佳比对。
序列的比较和两个序列之间百分比同一性的确定可以使用数学算法来完成。例如,两个氨基酸序列之间的百分比同一性可以通过以下方式来确定:使用Needleman和Wunsch((1970)J.Mol.Biol.48:444-453)算法,该算法已并入到GCG软件包(可在www.gcg.com上获得)的GAP程序中;使用默认参数,例如,Blossum 62打分矩阵,其空位罚分为12,空位扩展罚分为4,并且移码空位罚分为5。
方法
在临床环境中,可以通过多种方法中的任何一种将载体引入到受试者中,其中每种方法都是本领域熟悉的。尽管可以使用其他方法,但在一些实施方案中,将基因疗法载体递送至视网膜的选择途径是经由视网膜下注射。这提供了进入视网膜的RPE和光感受器细胞的通道。动物研究示出,在视网膜下注射之后,不同血清型的AAV可以有效地转导这些细胞群体(Vandenberghe等人,PLoS One.2013;8:e53463.PMCID:3559681;Vandenberghe andAuricchio,Gene Therapy.2012;19:162-8;Vandenberghe等人,Science translationalmedicine.2011;3:88ra54;Dinculescu等人,HumGene Ther.2005;16:649-63;Boye等人,Mol Ther.2013;21:509-19;Alexander and Hauswirth,Adv Exp Med Biol.2008;613:121-8)。视网膜下注射方案正用于正在进行的基因增补疗法临床试验,该疗法用于由RPE65和CHM基因遗传性疾病的突变引起的视网膜变性(Maguire等人,New England Journal ofMedicine.2008;358:2240-8;Bainbridge等人,New England Journal of Medicine.2008;358:2231-9;Cideciyan等人,Proceedings National Academy Sciences USA.2008;105:15112-7;Maguire等人,Lancet.2009;374:1597-605;Jacobson等人,ArchivesOphthalmology.2012;130:9-24;Bennett等人,Science translational medicine.2012;4:120ra15;Allocca等人,Lancet.2014;383:1129-37)。可以使用标准手术方案进行视网膜下注射(例如,如以下文献中所描述的:Maguire等人,2008同上;Bainbridge等人,2008同上;Cideciyan等人,2008同上;MacLaren等人,2014同上)。
受试者
本方法可以用于治疗患有NMNAT1相关视网膜病变/视网膜变性的受试者,例如Leber先天性黑蒙或早发型严重视网膜营养不良(EOSRD)。此类受试者可以通过本领域技术人员来鉴定并且通过基因测试来确认诊断(例如,测序以鉴定受试者的NMNAT1基因中突变的存在)。参见,例如,Kumaran等人,“Leber Congenital Amaurosis/Early-Onset SevereRetinal Dystrophy Overview”于Gene Reviews,Adam MP,Ardinger HH,Pagon RA等人编.Seattle(WA):University of Washington,Seattle;1993-2020;Kumaran等人,RetinCases Brief Rep.2018Jul 11.doi:10.1097/ICB.0000000000000754。
实施例
在以下实施例中进一步描述了本发明,这些实施例不限制权利要求书中所描述的本发明的范围。
材料和方法
在以下实施方案中使用以下材料和方法。
小鼠系
p.V9M-Nmnat1小鼠系先前衍生自N-乙基-N-亚硝基脲(ENU)诱变筛选25。出于增加繁殖力的目的,使原始C57Bl/6J系与野生型129S6/SvEvTac小鼠(Taconic,Rensselaer,NY)和野生型C57BL/6J小鼠(The Jackson Laboratory,Bar Harbor,ME)进行交替异型杂交,以保持混合C57Bl/6J-129S6遗传背景。单独维持仅用于筛选试剂组分毒性的野生型CD1-IGS小鼠(Charles River,Wilmington,MA)。雄性和雌性小鼠无偏好地用于实验。
动物养殖
在薛本斯眼科研究所动物护理房(the Schepens Eye Research InstituteAnimal Care Facility)中饲养和维持小鼠,在该房中,对小鼠喂食4%脂肪啮齿动物饮食,并且随意饮水,并且放置在12小时光照/12小时黑暗循环中。本研究符合《视觉和眼科研究协会关于在眼科和视觉研究中使用动物的声明(the Association for Research inVision and Ophthalmology Statement for the Use of Animals in Ophthalmic andVision Research)》,并且所有程序都得到了薛本斯眼科研究所动物护理和使用委员会(the Animal Care and Use Committee of the Schepens Eye Research Institute)的批准。
基因分型
根据制造商说明书,使用Allele-In-One Mouse Tail Direct Lysis Buffer(Allele Biotech,San Diego,CA)制备用于聚合酶链式反应(PCR)的组织生检。使用正向引物5’-CATGGCTGTGCTGAGGTG-‘3(内含子1;SEQ ID NO:4)和反向引物5’-AACAGCCTGAGGTGCATGTT-‘3(外显子2;SEQ ID NO:5)进行PCR,以扩增包括密码子9的Nmnat1的691bp区域。20μL PCR反应的最终浓度为每种引物200μmol/L、每种dNTP(dATP、dGTP、dTTP和dCTP)200nmol/L、2mmol/L MgCl2和1单位Hot FirePol DNA聚合酶(Solis BioDyne,Tartu,Estonia)。热循环方案为95℃达14分钟;95℃达45秒、53℃达45秒、72℃达30秒的30个循环;72℃达7分钟。接下来,使用引物5’-ACGTATTTGCCCACCTGTCT-‘3对扩增产物进行Sanger测序;SEQ ID NO:6;并且在c.25处分析电泳图,以将每只小鼠鉴定为针对Nmnat1V9M而言野生型、杂合或纯合的。
DNA构建体和AAV载体制备
将由DNA 2.0(Menlo Park,CA)设计并合成到gBlock基因片段(Integrated DNATechnologies,Coralville,IA)中的密码子优化的人NMNAT1 cDNA(图6A)并入到构建体中,然后将该构建体包装到重组AAV病毒载体中。
使用标准无内毒素分子克隆技术生成含有完整构建体的质粒,并且通过对NMNAT1和跨越连接位点的区域进行测序来验证。
如先前所描述的34,AAV由马萨诸塞州眼耳Grousbeck基因疗法中心(theGrousbeck Gene Therapy Center of Massachusetts Eye and Ear)制备。将纯化病毒收集在含有1x PBS、35mM NaCl和0.001%Pluronic F68表面活性剂的最终缓冲液中,然后进行滴定。如果需要,则使用相同的缓冲液来进一步稀释病毒,以达到靶剂量。为了协助注射程序,将<0.25%的荧光素(AK-Fluor,Akorn,Lake Forest,IL)混合到工作溶液中作为示踪剂。
为了排除AAV2/Anc80试剂毒性,使用特定修改进行注射:1)仅在产生后添加表面活性剂的情况下制造AAV2/Anc80,2)将AAV2/Anc80稀释在低盐稀释缓冲液中,3)仅盐水(载体去除),4)盐水加100x浓度的表面活性剂(去除载体),5)具有荧光素的盐水(载体去除)。
全身麻醉
对于全身麻醉,通过腹膜内注射递送氯胺酮/甲苯噻嗪(xylazine)的混合物。两周龄小鼠接受37.5mg/kg氯胺酮和3.8mg/kg甲苯噻嗪的剂量,并且成年小鼠接受100mg/kg氯胺酮和20mg/kg甲苯噻嗪的剂量。为了抵消永久性麻醉诱导的角膜混浊的形成,在使用氯胺酮/甲苯噻嗪的每次恢复程序(即AAV注射、体内成像、ERG)之后,立即通过皮下注射施用2mg/kg剂量的育亨宾(Yohimbine)HCL(Wedgewood Pharmacy,Swedesboro,NJ)。51,52对于新生小鼠,通过间接暴露于冰来诱导低体温全身麻醉。53
病毒递送
在两周龄小鼠中,使用带有RPE套组(kit)的Micro4显微注射泵(World PrecisionInstruments,Sarasota,FL)来将病毒试剂递送到视网膜下空间或玻璃体腔中。使用托吡卡胺(Tropicamide)(1%)或托吡卡胺(0.25%)、盐酸去氧肾上腺素(0.25%)、环喷托酯(1%)的半混合物来对瞳孔进行扩张。用氯胺酮/甲苯噻嗪对小鼠进行深度麻醉,并且使用盐酸丙美卡因(Proparacaine hydrochloride)(0.5%)进行局部麻醉。接下来,将眼突出,并且使用30g注射器针刺入紧靠角膜缘巩膜外血管后部的颞上巩膜和视网膜,以便为33g钝端套管形成进入途径。
对于视网膜下注射,经由解剖显微镜,通过扩张的瞳孔观察套管穿过玻璃体腔的穿越情况,并且将套管尖端定位在眼的下鼻象限的后部部分的视网膜下空间中。连续注射四次185.5nL试剂推注(总共0.75μL),并且通过荧光素示踪剂增强的可视化来确认泡的形成。注射后将套管保持在原位大约三秒钟以避免试剂回流,然后轻轻地从眼中取出。最后,通过用棉签填塞来处理进入伤口。然后用人工泪液(Blink Tears,Abbott Laboratories,Chicago,IL)给眼补水,并且小鼠在加热垫上从麻醉中恢复。
两周龄小鼠的玻璃体内注射程序是相同的,不同之处在于,在注射期间将套管尖端定位在玻璃体腔的中心。
在新生儿中,使用FemtoJet 4i显微注射系统(Eppendorf,Hamburg,Germany)进行视网膜下注射。当将小鼠在冰上进行麻醉时,使用30g皮下注射器针的尖端将上眼睑和下眼睑分开。将眼突出,并且将定制斜面玻璃针(目录号C060609,Origio,Trumbull,CT)通过巩膜直接插入并定位在下方的视网膜下空间中。在330hPa的压力下,在6秒内施用0.5μL试剂的单次推注,然后将针保持在原位大约三秒以避免回流,然后轻轻取出。小鼠在加热垫上从麻醉中恢复。
体内视网膜成像
如先前所描述的25,使用眼底摄影法和频域光学相干断层扫描技术(OCT)获取视网膜的正面(en face)和横截面图像(图7)。眼底摄影法程序的一个添加项是,一些图像是通过过滤器拍摄的,该过滤器允许EGFP可视化,并且因此允许对注射质量进行早期评价。此外,在跨越视网膜的多个位置处进行矩形体积扫描,以便可以准确测量上区域和下区域,并且对十次扫描进行配准/取平均以生成最终图像。使用InVivoVue OCT软件(Bioptogen),从外丛状层至视网膜色素上皮进行四次大约等距的卡尺测量,以测量光感受器层厚度。
视网膜电描记术
如先前所描述的25,从小鼠收集全视野闪光(flash)视网膜电流图。简而言之,使小鼠暗适应过夜,并且分别使用0.01cd.s/m2(暗视)和10cd.s/m2(暗视)宽带光刺激产生视杆细胞(rod)和混合视杆细胞/视锥细胞响应。接下来,通过暴露于稳定的30cd/m2(明视)宽带光10分钟来使小鼠进行光适应,并且在获得对20cd.s/m2(明视)宽带光刺激的视锥细胞分离的响应期间,在背景中保持此光。
统计学
在Prism版本8.2.1(GraphPad,San Diego,CA)中完成统计分析。对于OCT和视网膜电流图(ERG)时间进程,使用混合效应回归模型进行双向ANOVA。在分析治疗效果时,将未被注射眼的下方视网膜用作阴性对照,将其与所有其他测量的平均值进行比较。Dunnett事后检验用于解释多项比较测试所产生的第I类误差。在分析注射效果时,所有定量数据均报告为平均值±S.E.M。
定制抗人NMNAT1抗体开发
Aves Labs(Tigard,OR)使用纯化全长人NMNAT1作为抗原在鸡中产生定制多克隆抗体。该抗体针对人NMNAT1反应强烈,与小鼠直系同源物的交叉反应性最小。在图8中,用DAPI复染的人源ARPE-19细胞54的细胞核示出了与NMNAT1定位一致的稳健抗体标记(顶部行)。在小鼠视网膜中,免疫荧光最小(中间行),除非用AAV处理,以使其表达人NMNAT1(底部行);在后者的情况下,免疫反应性较强并且定位于细胞核。
离体视网膜成像
免疫组织化学法
通过CO2窒息对小鼠实施安乐死,并且立即使用Masterflex蠕动泵(Cole-Parmer,Vernon Hills,IL)通过心脏进行灌注。每只动物首先用含有2U肝素/mL的0.13mol/L磷酸盐缓冲盐水(PBS)pH 7.2至7.4进行灌注,直到灌注液变得澄清,随后灌注约40mL的2%多聚甲醛(PFA)。两种溶液在灌注时均加热至约37℃。使用小容器烧灼器(#18000-00,FineScience Tools,Foster City,CA)标记紧邻上角膜缘前部的角膜。摘除眼,在室温下在2%PFA中孵育0.5小时,去除眼前节,然后将剩余的眼杯在室温下在2%PFA中再次孵育0.5小时,然后在室温下浸入30%蔗糖中至少1小时。如先前所描述的25,包埋眼杯,通过冷冻切片术以10μL厚度切片,免疫标记和染色,并且使用Eclipse Ti荧光显微镜(Nikon,Tokyo,Japan)在荧光或明视野模式下成像,除非在其它情况下用TCS SP8共聚焦显微镜(Leica,Wetzlar,Germany)成像。
苏木精和伊红标记
在通过CO2窒息进行安乐死之后,摘除眼,将其在PBS中洗涤,立即浸入O-fix组织固定剂[<70%乙醇、<5%甲醇、<7%乙酸、<4%乙酸和4%甲醛](Leica,3800676)中,并且在固定剂中孵育过夜。然后将样品在一系列分级乙醇中脱水,在二甲苯中澄清,并且包埋入石蜡(Paraplast Plus,McCormick Scientific,Richmond,IL)中。使用Leica RM2145切片机(Leica Biosystems,Buffalo Grove,IL)制备6μm厚切片,然后用Gill's#2Hematoxylinand Eosin-y(Fisher Scientific,Pittsburgh,PA USA)对载玻片进行染色,然后在用Permount封固介质的情况下盖上盖玻片。
实施例1.DNA构建体和AAV试剂制备
将密码子优化的人NMNAT1 cDNA(图1A)并入到构建体中,然后将该构建体包装到重组AAV病毒载体中。已发现,密码子优化改善人类基因在转导细胞中表达的水平和持续时间,而不改变蛋白质产物的氨基酸序列26-30。同样地,并入到840bp NMNAT1 cDNA中的所有174个核苷酸取代都是沉默的,从而定义了正常的人蛋白质序列。
作为本研究的一部分,组合使用四种DNA构建体和六种AAV载体亚型以产生四种试剂,将这些试剂递送至小鼠。将NMNAT1由泛表达CASI启动子驱动的构建体包装到AAV2/9的自身互补(SC)和单链(SS)版本二者中。选择SC载体进行测试是因为其比传统的SS载体更快地激活基因表达。31此外,将含有EGFP(增强型绿色荧光蛋白)报告基因的构建体32包装到SS.AAV2/9中,该基因也由CASI启动子驱动并且随后是WPRE(土拨鼠肝炎病毒翻译后调控元件),其用于增强小鼠视网膜中AAV2介导的转导。在将SC.AAV2/9试剂(图1B)和SS.AAV2/9试剂(图1C)以每微升1x108个基因组拷贝(gc/μL)加入该EGFP试剂中之后,立即将其递送至小鼠,以便可以通过体内和离体成像确认基因表达。制作了另一构建体,其中NMNAT1由泛表达CAG启动子驱动并随后是T2A切割序列、EGFP、然后是WPRE。在细胞中翻译后,在T2A切割位点处酶促分离NMNAT1-EGFP融合蛋白,33以避免破坏标称蛋白质构象和动力学。将该构建体包装到AAV2/Anc80中,这是一种合成AAV2,通过定向进化产生以规避先天性免疫,并且已显示为可以在小鼠中有效地转导视网膜细胞(图1D)。34最后,将上文描述的相同CASI.NMNAT1构建体包装到AAV2/7m8载体中,与其他载体不同,该载体可以通过玻璃体内注射递送至所有视网膜层(图1E)。35
实施例2.使用自身互补载体的基因增补保留了视网膜结构
使用自身互补(SC)AAV2/9(SC.AAV2/9)试剂对两周龄的Nmnat1V9M/V9M小鼠施用基因增补疗法,以剂量依赖性方式稳定地保留了Nmnat1V9M/V9M小鼠的视网膜结构。这一发现是通过使用光学相干断层扫描技术(OCT)在体内收集的光感受器层厚度测量确定的。对于被注射的眼,靠近下方视网膜的注射位点进行测量,预期此处的拯救是最稳健的,并且在远离注射位点的上方视网膜中进行测量。在Nmnat1V9M/V9M小鼠的未被注射同类眼中和在年龄匹配同窝对照的未被注射眼中的测量是在下方视网膜中、在与被注射眼中进行测量的平面等效的平面中获得的(图2)。
分别为1x108(gc/μL)和2x109gc/μL的中滴度注射和高滴度注射均在被注射的视网膜上提供了显著的拯救(图3A,顶部行)。例如,在九月龄时,与同类未被注射眼的31.3μm±0.8厚的下方区域相比,注射2x109gc/μL剂量的眼分别具有100.0μm±4.8(68.7μm差异,p<0.0001)和96.1μm±6.4(64.8μm差异,p<0.0001)的下方和上方光感受器层厚度。处理的视网膜通常在下方野生型视网膜的厚度的约20%范围内。在2x109gc/μL剂量后,下方视网膜(靠近注射位点)与上方视网膜(远离注射位点)之间的光感受器层厚度趋向于相似(p>0.05)。然而,在1x108gc/μL剂量后,由于与注射部位的距离,视网膜厚度降低了约20%。本发明人不能测试大于2x109gc/μL的剂量是否会提供额外的优势,因为这是全强度制剂。
在1x107(gc/μL)(该试剂的最低测试剂量)下,与同类未被注射视网膜相比,在Nmnat1V9M/V9M视网膜的下方区域(靠近注射位点)观察到光感受器层厚度的适度拯救。这一发现仅在四个月和六个月时达到统计显著性,示出了与同类视网膜下方区域的差异分别为25.8μm±6.5(p=0.014)和30.2μm±7.9(p=0.035)。所拯救区域的光感受器层仅具有未被注射野生型同窝对照视网膜等效区域的厚度的大约一半,并且在任何时间点,远离注射位点的视网膜区域(即上方视网膜)都没有示出拯救的证据。
由于NMNAT1活性的丧失可能因突变而有很大不同,因此了解NMNAT1是否过表达是重要的。出于这一目的,对野生型小鼠注射了与突变同窝仔相同滴度的SC.AAV2/9试剂。在所有剂量和时间点上,光感受器层厚度通常不受影响,仅在三个情况中注意到具有统计显著性的差异(图3A,底部行)。
最大的差异是在2x109gc/μL注射后九个月时经注射的视网膜的上方区域中,其中测量到17.9μm±6.9的降低(p=0.039)。然而,考虑到同一视网膜的下方区域(靠近注射部位)未受影响,减量不可能是由于毒性。
实施例3.单链载体不能拯救疾病表型
与SC.AAV2/9试剂相比,SS.AAV2/9、AAV2/7m8和AAV2/Anc80试剂不能为Nmnat1V9M /V9MM视网膜提供稳定的结构拯救。在两月龄时,与未被注射同类眼的相同区域相比,注射SS.AAV2.9试剂的突变视网膜的下方区域示出了非常适度的11.4μm±4.3(p=0.033)拯救(图3B),并且AAV2/7m8试剂没有效果。5.5x108gc/μL剂量的AAV2/Anc80试剂在突变视网膜中产生了短暂的拯救,该拯救未持续超出两个月龄,并且限制在下方区域。在1.5和2个月的时间点,AAV2/Anc80处理的视网膜的下方区域的光感受器厚度比未被处理同类视网膜的光感受器厚度分别大42.0μm±3.7(p=0.0005)和27.5μm±9.9(p=0.045)(图3C)。当剂量降低到1x108gc/μL时没有观察到拯救,并且将AAV2/Anc80试剂剂量增加到≥1x109gc/μL由于毒性而失败。在野生型和NMNAT1V9M/V9M视网膜二者中,AAV2/Anc80试剂的高滴度注射导致视网膜变性,出现玻璃体内细胞浸润物36,37以及六周龄时严重的视网膜脱离(图4)。为了测试与AAV2/Anc80试剂一起使用的稀释缓冲液的组分是否有助于这一结果,将该溶液的组分在不同条件下注射到野生型小鼠中。调节表面活性剂、盐和荧光素浓度不导致通过OCT或组织学可检测到的视网膜损伤(数据未示出)。
如通过OCT所观察到的,与SC.AAV2试剂相关的NMNAT1V9M/V9M视网膜的结构拯救是通过光学显微术离体证实的。苏木精和伊红(H&E)染色证实,与未被处理的突变视网膜形成鲜明对比,所拯救视网膜的良好转导区域具有完整的所有细胞类型,并且具有复杂的光感受器外段(图5A)。然而,被处理的突变视网膜的视网膜层似乎比未被处理的野生型视网膜略薄,这与来自OCT图像的前述测量一致。虽然被注射的野生型视网膜通常与未被注射的同类视网膜难以区别,但存在着光感受器外段部分缩回且其他视网膜层更薄的区域。免疫标记实验示出,在被注射的突变型和野生型视网膜二者中,几乎每种类型的视网膜细胞的细胞核中都可检测到转基因表达(图5B)。
此外,泛视网膜转基因表达通常延伸超出组织的三分之二(图3B)。使用与小鼠NMNAT1具有最小交叉反应性的定制鸡多克隆α-人NMNAT1抗体来完成免疫标记(图6)。
实施例4.使用自身互补载体的基因增补保留了视网膜功能
为了评估视网膜的结构拯救是否转换为功能的保留,通过ERG在体内测量对光刺激的视杆细胞、混合视杆细胞/视锥细胞和视锥细胞分离的响应,所述ERG是一种用于测量视网膜对光刺激的电响应的非侵入性程序。具体而言,将由ON双极细胞介导的ERG b波的量级38,39用作光感受器响应的间接指标40以及内层视网膜功能的测量。对于用SC.AAV2/9试剂处理的视网膜,到四月龄时,b波显著大于未被处理同类眼的b波,并且对于明视刺激,到六月龄时仍然如此,并且对于暗视和混合视杆细胞/视锥细胞刺激,到至少九个月仍然如此。在九月龄时,被处理的突变视网膜中的明视ERG倾向于比未被处理的同类视网膜中的明视ERG更大(图7A,顶部行)。例如,在六月龄时,对于每种条件,被处理的突变视网膜的响应都超过了同类视网膜的响应:视杆细胞响应为156.1μV±23.4对比36.0μV±7.1(p=0.0013),混合视杆细胞/视锥细胞响应为293.6μV±51.1对比62.0±11.8(p=0.0034),以及视锥细胞响应为101.3μV±18.6对比25.3±6.2(p=0.0067)。
此外,与拯救相关的响应始终低于未被处理野生型视网膜的响应。这种效果可能源于两个来源。首先,OCT和组织学图像示出,即使是最稳健的结构拯救也是不完整的。
其次,注射本身似乎抑制ERG信号,因为被注射野生型视网膜产生的ERG的振幅倾向于小于同类视网膜的振幅。
不管怎样,对于每种刺激条件,所拯救ERG波形的结构和隐含时间(implicittime)都是正常的(图7A,底部行)。1x108gc/μL的SC.AAV2试剂在被注射的突变视网膜中示出了拯救的证据(图7B,顶部行),而在存在结构保留的两月龄时没有观察到AAV2/Anc80试剂的拯救迹象(图7B,底部行)。
实施例5.自身互补载体比单链载体更早激活NMNAT1表达
为了理解为何当经由SC.AAV2/9载体递送转基因时视网膜结构和功能得到更大程度的保留,本发明人对早期NMNAT1表达谱进行了表征。在注射后十四天,由SC.AAV2/9递送的NMNAT1的免疫标记在所有视网膜层中都可检测到,其中在外核层(ONL)中密度特别高(图8A)。相反,在SS.AAV2/9和AAV2/Anc80注射的视网膜中,NMNAT1的检测是稀疏的,标记的光感受器相对较少,即使SS.AAV2/9载体携带相同的货物并且AAV2/Anc80载体已显示以宽覆盖转导小鼠视网膜感光器。34无论使用何种递送载体,在注射后七天检测不到NMNAT1。
实施例9.成功的干预需要光感受器的转导
此外,为了确定较早期干预是否会从AAV2/Anc80试剂中产生更好的功效,在出生后第0天(P0)到P2对新生小鼠进行注射。虽然大多数细胞类型示出了强烈的NMNAT1表达,包括视锥细胞光感受器,但视杆细胞光感受器中的表达非常弱(图8B);视锥细胞光感受器以其细胞核的位置在ONL的最外行中区分。41相似地,在两周龄小鼠中,在经由AAV2/7m8玻璃体内递送NMNAT1后四周,通过OCT未观察到光感受器层的保留,并且通过免疫组织化学确定,尽管具有强烈的内层视网膜信号,但外层视网膜中不存在NMNAT1表达(图8C)。
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其他实施方案
应当理解,虽然已经结合本发明的详细描述描述了本发明,但是前述描述旨在说明而不是限制本发明的范围,本发明的范围由所附权利要求书的范围限定。其他方面、优点和修改在所附权利要求书的范围内。
Claims (16)
1.治疗人受试者中由烟酰胺单核苷酸腺苷酰转移酶1(NMNAT1)基因中的突变引起的视网膜变性的方法,所述方法包括将治疗有效量的腺相关病毒(AAV)载体递送至所述受试者的眼,所述载体包含编码人NMNAT1的序列,所述序列与在视网膜细胞中,优选在光感受器中驱动表达的启动子可操作地连接。
2.根据权利要求1所述的方法,其中所述启动子是CAG、CASI、CMV、RHO或视紫质激酶(GRK1)启动子。
3.根据权利要求2所述的方法,其中所述编码NMNAT1的序列与野生型(SEQ ID NO:2)或密码子优化的(SEQ ID NO:1)序列至少80%相同。
4.根据权利要求1至3所述的方法,其中所述载体经由视网膜下注射递送。
5.增加人受试者的眼中NMNAT1的表达的方法,所述方法包括将治疗有效量的腺相关病毒2型(AAV2)载体递送至所述受试者的眼,所述载体包含编码人NMNAT1的序列,所述序列与在视网膜中,优选在光感受器细胞中驱动表达的启动子可操作地连接。
6.根据权利要求5所述的方法,其中所述启动子是CAG、CASI、CMV、RHO或视紫质激酶(GRK1)启动子。
7.根据权利要求5所述的方法,其中所述NMNAT1序列是密码子优化的。
8.根据权利要求5至7所述的方法,其中所述载体经由视网膜下注射递送。
9.腺相关病毒2型(AAV2)载体,所述载体包含编码人NMNAT1的序列,所述序列与在视网膜中,优选在光感受器细胞中驱动表达的启动子可操作地连接。
10.根据权利要求9所述的载体,其中所述启动子是CAG、CASI、CMV、RHO或视紫质激酶(GRK1)启动子。
11.根据权利要求9所述的载体,其中所述NMNAT1序列是密码子优化的。
12.包含根据权利要求9至11所述的载体的药物组合物,所述药物组合物被配制成用于经由视网膜下注射递送。
13.根据权利要求9至11所述的载体,所述载体用于治疗人受试者的眼中由NMNAT1中的突变引起的视网膜变性。
14.根据权利要求9至11所述的载体,所述载体用于增加人受试者的眼中NMNAT1的表达。
15.根据权利要求12所述的药物组合物,所述药物组合物用于治疗人受试者的眼中由NMNAT1中的突变引起的视网膜变性。
16.根据权利要求12所述的药物组合物,所述药物组合物用于增加人受试者的眼中NMNAT1的表达。
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
US202062988260P | 2020-03-11 | 2020-03-11 | |
US62/988,260 | 2020-03-11 | ||
PCT/US2021/021936 WO2021183779A1 (en) | 2020-03-11 | 2021-03-11 | Gene therapy for nmnat1-associated retinal degeneration |
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KR20220152550A (ko) | 2022-11-16 |
CA3174781A1 (en) | 2021-09-16 |
WO2021183779A1 (en) | 2021-09-16 |
AU2021236233A1 (en) | 2022-09-22 |
EP4117644A1 (en) | 2023-01-18 |
MX2022011177A (es) | 2022-12-13 |
US20210299277A1 (en) | 2021-09-30 |
BR112022017970A2 (pt) | 2022-10-25 |
JP2023517929A (ja) | 2023-04-27 |
IL296262A (en) | 2022-11-01 |
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