CN104487579B - 使用aav衣壳变异体的高度有效的基因转移的组合物和方法 - Google Patents
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Abstract
本发明公开了用于AAV介导的基因治疗的改进的组合物和方法。
Description
本申请要求分别于2012年4月18日和2013年3月15日提交的美国临时专利申请第61/635,273和61/794,995号的优先权,各专利申请的全部内容以似乎完整地陈述的参考方式并入本文中。
技术领域
本申请涉及基因治疗和分子生物学领域。更具体地,本发明提供包含蛋白质衣壳变异体的AAV载体,该蛋白质衣壳变异体提高包含治疗上有利的转基因的AAV载体的转导效率。
背景技术
本说明书的全文中引用若干公开物和专利文件,以便描述本发明所属领域的状态。这些引用文件中的每一个以似乎完整地陈述的参考方式并入本文中。
腺相关病毒(AAV)的一种小的(20nm)复制缺陷的无包膜病毒。已在人和非人灵长类动物中鉴定出许多不同的AAV血清型。AAV基因组是由在两端具有145bp的末端反向重复(ITR)的单链DNA所构成。存在两个开放阅读框(ORF):Rep和Cap。虽然Rep产物对于AAV复制是必不可少的,但有3种衣壳蛋白(VP1、VP2、和VP3)是由Cap基因表达。VP1、VP2和VP3以1:1:10的比率共同地形成二十面体衣壳(Xie Q et al,2002)。在重组AAV(rAAV)载体产生期间,将由ITRs旁侧(flank)的表达盒被包装入AAV衣壳中。AAV的复制所需的基因不包含在该表达盒中。重组AAV被认为是最安全的,并且是最广泛使用的用于体内基因转移的病毒载体之一。这些载体可以感染来自于提供强的和持续的转基因表达的多种组织类型的细胞。它们也是非致病的,并且具有低的免疫原性(High KA,2011)。
基因治疗试验的一个直接目的是优化载体以在使载体剂量最小化的同时使组织转导最大化。当进入细胞时,AAV衣壳蛋白经历蛋白酶体介导的降解。AAV衣壳的表面暴露酪氨酸残基的磷酸化代表了经由泛素-蛋白酶体途径导致病毒降解的第一步骤中的一个步骤(Zhong L et al,2007)。细胞中大部分的受控蛋白水解是经过此途径而发生。泛素是在所有真核细胞中均可以发现的小蛋白质(约8.5kDa)。泛素连接到底物蛋白氨基酸的侧链。在其它泛素蛋白质经由最初连接的泛素连接到底物之后,形成多聚泛素链并且标记底物用于降解(Thrower JS et al 2000,Peng J et al 2003,Bedford L et al,2011)。已证明表面暴露酪氨酸残基的突变导致AAV 2载体的转导效率的提高(Zhong L et al,2008)。最近,若干个研究小组已证明该方案也可有效地用于在若干组织中的其它AAV血清型,包括AAV血清型6和8。
显然,在本领域中对于在有需要的患者中改善携带临床上重要的转基因的AVV的转导的组合物和方法存在着需求。
发明内容
根据本发明,提供了新型的腺相关病毒变异体,该变异体当与缺乏本文中公开的修饰的AAV血清型(例如,AAV1、AAV2、AAV8、AAV-rh74)相比时显示提高的转导效率。这种改进的载体可用于包括肝脏、肌肉、大脑和视网膜在内的多种组织的转导。
在一个实施方式中,提供腺相关病毒(AAV)载体,该载体包含改变的VP1衣壳蛋白,该改变的VP1衣壳蛋白包含赖氨酸残基替换(substitution),由此降低衣壳的泛素化并提高将变异体AVV转导入靶组织和细胞中的转导效率。在一个实施方式中,载体还包含异源核酸(例如,包含AAV末端反向重复和异源核酸序列的小基因),该异源核酸可操作地连接到调控序列,该调控序列引导宿主细胞中由异源核酸序列对产物的表达。在一个优选的实施方式中,AAV载体包含如在本文中所给出的表中提供的VP1中的一个或多个赖氨酸替换。在另一个实施方式中,AAV载体属于AAV8血清型,并且含有表3中示出的改变。
在一个优选实施方式中,包含变异体衣壳蛋白的本发明的AAV载体可用于治疗性肽或者治疗性核酸的表达。这种肽类包括但不限于:抗病毒RNAi分子、第八因子、第九因子或者其功能性片段。其它的表达产物包括例如:IgG、IgM、IgA、IgD、IgE、嵌合免疫球蛋白、人源化抗体、或者单链抗体。在一个方面,表达产物是可用于抑制HCV(丙型肝炎病毒)感染和复制的RNAi。在另一个实施方式中,表达产物是可用于下调所研究的靶细胞的反义核酸。
在本发明的另一个实施方式中,提供包含在生物学相容载体中的本发明的变异体AAV载体的药物组合物。本发明还涉及包含本文中所公开载体的细胞培养物。
本发明还涉及一种将转基因传递至受试对象中的细胞的方法;所述方法包括如本文中所公开的使细胞与AAV载体接触的步骤,其中所述AAV载体包含转基因,其中所述载体中的VP1衣壳序列中的赖氨酸替换(substitution)与降低的泛素化以及提高的转导效率有关。
在最后的方面,本发明提供诱饵病毒变异体,该诱饵病毒变异体不足以感染细胞,但可有效地阻断携带有利的转基因的病毒变异体的抗体中和,原因在于这两种病毒变异体的结构相似性。用于此目的的示例性衣壳变异体包括例如K38R、K143R、K510R和K709R。
附图说明
图1:在这里所使用的AAV1、AAV2和AAV8的表面上的赖氨酸:所使用的AAV血清型的PDB数量如下:AAV1:3NG9、AAV2:1LP3、和AAV8:2QA0。箭头代表各自的赖氨酸残基。(A)在AAV1 VP3的表面上有11个赖氨酸。残基的颜色如下:K258红、K459蓝、K491黄、K493品红、K508青、K528深肉色、K533淡绿、K545淡蓝(蓝灰色)、K567深肉色、K666淡青、K707灰。(B)在AAV2 VP3的表面上有10个赖氨酸。残基的颜色如下:K258红、K490黄、K507青、K527深肉色、K532淡绿、K544淡蓝(蓝灰色)、K549淡黄、K556浅品红、K665淡青、K706灰。(C)在AAV8 VP3的表面上有8个赖氨酸。残基的颜色如下:K259红、K333绿、K510青、K530深肉色、K547淡蓝(蓝灰色)、K569深肉色、K668淡青、K709灰。应注意AAV1的K567以及K528和AAV8的K530以及K569在结构中为并排的,并且显示具有相同的颜色。
图2:将AAV8衣壳的若干赖氨酸残基突变为精氨酸。在动物经过尾静脉注射病毒8周后从这些动物中采集血液。利用ELISA检测hF.IX水平(A)利用软件预测以高和中等置信度水平被泛素化的残基突变为精氨酸。经过尾静脉给每只小鼠注射2.5×1010个病毒颗粒(B、C)。利用软件预测以低置信度被泛素化的残基突变为精氨酸。经过尾静脉给每只小鼠注射2.5×109个病毒颗粒。(B)K569R和K668R衣壳突变(C)对K38R、K143R、K259R、K510R、K547R衣壳突变的影响与对K530R突变的影响进行比较。
图3:K向R衣壳突变的组合(A)K137R、K33R和K530R突变的组合仍然高于野生型,但在统计学上并非不同于K530R。(B)K709R突变对AAV8转导产生负面影响。将K709R突变加入到K(137/333/530)R突变体也降低病毒的转导。(C)多个赖氨酸-精氨酸突变的组合不提高转导率。(D)3个赖氨酸突变为精氨酸残基与4或6个酪氨酸突变为苯丙氨酸残基的组合降低转导率。
图4:AAV1转导:(A)在三个不同的MOI 5K、50K和500K处用AAV-1赖氨酸突变体转导的HHL5-B7肝细胞的CTL杀伤。将肽(用于AAV1的IPQYGYLTL;用于AAV2的VPQYGYLTL)用作阳性对照。LDH释放与细胞杀伤相关联。(B)在50K和500K MOI处的不同结构之间对GFP阳性细胞总数和GFP表达进行了比较。
图5:AAV2转导:(A)对AAV2 K137R、K527R或K532R突变体与WT AAV2在HHL5细胞系转导率方面进行了比较。在10K、50K、100K和500K MOI处转染这些细胞,并且在24小时后对GFP表达进行检查。(B)显示利用测试变异体的LDL释放所测量的细胞毒性的图。
图6:CTL测定,其中用AAV-2载体以增加的浓度将靶肝细胞进行转导,然后与HLA相配效应子细胞一起保温培养。如图所示的AAV载体编码的野生型AAV-2衣壳或者单个赖氨酸突变。针对AAV-2MHC I类表位VPQYGYLTL的效应子是从在体外扩大的PBMC中获得,并且效应子-靶比率为10:1,结果表示为相对于野生型载体的CTL活性的百分比(与用10%SDS处理的细胞进行比较的细胞毒性%,作为在背景减除后的最大细胞毒性对照)。
图7:RH74数据:利用对于人F.IX是特异性的ELISA所测量的血浆中人F.IX转基因表达水平。(A)TMR=K的定义(137/333/530)R,HMF的定义=Y(253/275/447/703/707/733)F。(B)HMR的定义:K(137/259/333/530/552/569)R,195/202的定义:G195A+L199V+S201P+G202N。虽然HMR+195/202、HMR+195/202+K(38)R、HMR+195/202+K(51)R、HMR+195/202+K(61)R、和HMR+195/202+K(77)R当给小鼠注射时产生较高的hFIX产量,但HMR+195/202+K(122/123)R或者HMR+195/202+K(142/413)R注射根本不产生任何可检测的hFIX。(C):与Rh74 WT相比RHM13_1突变体产生类似的hFIX水平,而来源于RHM17_1-处理的小鼠的hFIX水平仅仅超过背景值。(D)与Rh74WT相比,RHM14_2突变体产生类似的hFIX水平;RHM15_1的性能是在AAV8和AAVrh74 WT之间。
具体实施方式
根据本发明,我们已发现使AAV衣壳上的赖氨酸残基突变到精氨酸残基提高腺相关病毒转导效率。我们的初步实验证明预测是泛素化靶的赖氨酸残基的单个替换导致与接受未修饰的AAV载体的动物相比小鼠中较高水平的人第九因子IX(FIX)转基因的表达。本文中描述的AAV赖氨酸突变体可以用于有利于产生将肝脏、CNS、肌肉、和与野生型AAV衣壳相比具有较高效率的其它器官作为靶的载体。因此,此发现可以用于开发用于治疗血友病A和B、亨廷顿舞蹈病、和需要提高的将期望的转基因转导入所关注的靶组织中的转导水平的实际上任何其它疾病的治疗剂。
提供下面的定义,以便于对本发明的理解。
I.定义:
“基因治疗”是将基因***个体的细胞和/或组织中以治疗疾病,通常是遗传病,其中用功能性的等位基因来替换或补充缺陷突变体等位基因。
来自细小病毒科的“腺相关病毒”是具有单链DNA的基因组的小病毒。可以在染色体19上的特定位点处将遗传物质***这些病毒,并且这些病毒是优选的,因为它们与人中的致病性疾病不相关。
“治疗性”肽或蛋白质是可缓解或减轻由于细胞或受试对象中蛋白质缺乏或缺陷所造成的症状的肽或蛋白质。可替代地,“治疗性”肽或蛋白质是为受试对象赋予利益(例如抗癌作用)的肽或蛋白质。治疗性肽和蛋白质包括但不限于:CFTR(囊性纤维化跨膜调节蛋白)、肌养蛋白(包括肌养蛋白小基因的蛋白质产物,参见例如Vincent et al(1993)NatureGenetics 5:130)、肌营养不良相关蛋白(Tinsley et al(1996)Nature 384:349)、凝血因子(Factor XIII、Factor IX、Factor X等)、单克隆抗体(Lewis et al,2002)、***、LDL受体、脂蛋白脂肪酶、鸟氨酸氨甲酰基转移酶、β-珠蛋白、α-珠蛋白、血影蛋白、α-抗胰蛋白酶、腺苷脱氨酶、次黄嘌呤鸟嘌呤磷酸核糖转移酶、β-葡糖脑苷脂酶、鞘磷脂酶、溶酶体氨基己糖苷酶、支链酮酸脱氢酶、激素、生长因子(例如,***1和2、血小板衍生生长因子、表皮生长因子、神经生长因子、神经营养因子-3和-4、脑源性神经营养因子、胶质源性生长因子、α和β-转化生长因子等)、细胞因子(例如,α-干扰素、β-干扰素、γ-干扰素、白细胞介素-2、白细胞介素-4、白细胞介素-12、粒细胞-巨噬细胞集落刺激因子、淋巴毒素)、***基因产物(例如,单纯疱疹病毒胸苷激酶、胞嘧啶脱氨酶、白喉毒素、细胞色素P450、脱氧胞苷激酶、和肿瘤坏死因子)、赋予用于癌症治疗的药物耐药性的蛋白质、肿瘤抑制基因产物例如,p53、Rb、Wt-1、NF1、VHL、APC、等)、和在有需要的受试对象中具有治疗效果的任何其它肽或蛋白质。
其它示例性的治疗性肽或蛋白质包括可用于疾病治疗的治疗性肽或蛋白质,这些疾病包括但不限于:囊性纤维化(和其它肺病)、血友病A、血友病B、珠蛋白生成障碍性贫血、贫血症和其它血液病、艾滋病、阿尔茨海默氏病、帕金森氏病、亨廷顿舞蹈病、肌萎缩侧索硬化、癫痫、和其它神经***疾病、癌症、糖尿病、肌肉萎缩症(例如,Duchenne氏、Becker氏)、高雪氏病、赫尔勒氏病、腺苷脱氨酶缺乏症、糖原贮积病和其它代谢缺陷、视网膜退行性疾病(和其它眼病)、和实体器官的疾病(例如,大脑、肝脏、肾脏、心脏)。
本文中使用的术语“启动子”或“启动子”可以指代与编码重组产物的DNA序列相邻的DNA序列。优选地,启动子可操作地连接到相邻的DNA序列。与启动子不存在时所表达的重组产物的量相比较,启动子通常增加由DNA序列所表达重组产物的量。来自一个生物体的启动子可以用于提高来自来源于另一个生物体的DNA序列的重组产物表达。例如,脊椎动物启动子可用于脊椎动物中水母GFP的表达。另外,一个启动子元件可以增加为多个串联连接的DNA序列所表达的重组产物的量。因此,一个启动子元件可以增强一个或多个重组产物的表达。多个启动子元件对于本领域技术人员是众所周知的。
在一个实施方式中,高水平的组成型表达将是期望的。这种启动子的实例包括但不限于:逆转录劳斯肉瘤病毒(RSV)LTR启动子/增强子、巨细胞病毒(CMV)即刻早期启动子/增强子(见例如,Boshart et al,Cell,41:521-530(1985))、SV40启动子、二氢叶酸还原酶启动子、细胞质β-肌动蛋白启动子、和磷酸甘油激酶(PGK)启动子。
在另一个实施方式中,诱导型启动子会是理想的。诱导型启动子是由外来提供的化合物所调节的那些诱导型启动子,该外源性化合物是采用顺式或反式的形式,包括但不限于:锌诱导羊金属硫蛋白(MT)启动子、***(Dex)诱导的小鼠乳腺肿瘤病毒(MMTV)启动子、T7聚合酶启动子***(WO 98/10088)、四环素阻遏***(Gossen et al,Proc.Natl.Acad.Sci.USA,89:5547-5551(1992));四环素诱导***(Gossen et al,Science,268:1766-1769(1995);也见Harvey et al,Curr.Opin.Chem.Biol.,2:512-518(1998));RU486诱导***(Wang et al,Nat.Biotech.,15:239-243(1997)和Wang et al,Gene Ther.,4:432-441(1997)];和雷帕霉素诱导***(Magari et al,J.Clin.Invest.,100:2865-2872(1997);Rivera et al,Nat.Medicine.2:1028-1032(1996))。可用于本发明的其它类型的诱导型启动子是由特定生理状态(例如温度、急性期)所调节,或者仅仅用于复制细胞。
在另一个实施方式中,将使用用于所研究的转基因或核酸序列的天然启动子。当理想的是转基因或核酸序列的表达应当模拟天然表达时,天然启动子可以是优选的。当必须暂时地或进展地、或者以组织特异性方式、或者响应于特定的转录刺激来调节转基因或其它核酸序列的表达时,可使用天然启动子。在另一个实施方式中,也可将其它天然表达的控制元件,诸如增强子元件,多聚腺苷酸化位点或者Kozak共有序列用于模拟天然表达。
在一个实施方式中,重组病毒基因组包含可操作地连接到组织特异性启动子的转基因。例如,如果骨骼肌中的表达是期望的,则可使用在肌肉中起作用的启动子。这些包括:来源于编码骨骼α-肌动蛋白、肌球蛋白轻链2A、肌养蛋白、肌肉肌酸激酶、以及活性高于天然存在启动子的合成肌肉启动子的基因的启动子。见Li et al,Nat.Biotech.,17:241-245(1999)。已知用于肝白蛋白的组织特异性的启动子的实例,Miyatake et al.J.Virol.,71:5124-32(1997);乙型肝炎病毒核心启动子,Sandig et al,Gene Ther.3:1002-9(1996);甲胎蛋白(AFP),Arbuthnot et al,Hum.Gene Ther.,7:1503-14(1996)],骨骼(骨钙蛋白,Stein et al,Mol.Biol.Rep.,24:185-96(1997);骨唾液蛋白,Chen et al,J.BoneMiner.Res.11:654-64(1996))、淋巴细胞(CD2,Hansal et al,J.Immunol.,161:1063-8(1998);免疫球蛋白重链;T细胞受体a链)、神经元(神经元特异性烯醇化酶(NSE)启动子,Andersen et al。Cell.Mol.Neurobiol.,13:503-15(1993);神经丝轻链基因,Piccioli etal,Proc.Natl.Acad.Sci.USA,88:5611-5(1991);神经元特异性vgf基因,Piccioli et al,Neuron,15:373-84(1995)];等等。
本文中使用的术语“多个增强子”或“增强子”可以是指与编码重组产物的DNA序列相邻的DNA序列。增强子元件通常位于启动子元件的上游或者可以位于DNA编码序列的下游或者其内部(例如,被转录或翻译入重组产物中的DNA序列)。因此,增强子元件可以定位在编码重组产物的DNA序列的上游或下游的100个碱基对、200碱基对、或300个以上的碱基对的位置。增强子元件可以将由DNA序列所表达重组产物的量增加到超过由启动子元件所提供的增加的表达。本领域技术人员可容易地获得多个增强子元件。
本文中使用的“核酸”或“核酸分子”是指任何单链或者双链的DNA或RNA分子;如果是单链的,其互补序列的分子采用线性形式或环形形式。在对核酸分子进行描述中,在本文中可按照提供在5'到3'方向上的序列的正常惯例对特定核酸分子的序列或结构进行描述。关于本发明的核酸,有时使用术语“分离的核酸”。此术语,当应用于DNA时,是指在生物体的天然存在的基因组中紧密邻接的序列中所分离的DNA分子。例如,“分离的核酸”可包含被***载体(诸如质粒或病毒载体)中、或者结合入原核细胞或真核细胞或者宿主生物体的基因组DNA的DNA分子。
“载体”是指可以连接另一个基因序列或元件(DNA或者RNA)从而引起所连接序列或元件的复制的复制子,诸如质粒、黏粒、杆粒、噬菌体或病毒。
“表达操纵子”是指可具有转录和翻译控制序列并且促进宿主细胞或生物体中的多肽编码序列的表达的核酸片段,诸如启动子、增强子、转录起始信号(例如,ATG或AUG密码子)、多聚腺苷酸化信号、终止子等。
术语“转化”、“转染”、“转导”应是指将核酸导入细胞或宿主生物体的任何方法或手段,并且可以互换地使用以传达相同的含义。这种方法包括但不限于:转染、电穿孔、显微注射、感染、PEG融合等。
可以或者可以不将所导入的核酸结合(共价连接)入受体细胞或生物体的核酸。在细菌、酵母菌、植物和哺乳动物的细胞中,例如,可以将所导入的核酸作为附加体元件或者独立的复制子(诸如质粒)而维持。可替代地,所导入的核酸可以变得结合入受体细胞或生物体的核酸并稳定地维持在细胞或生物体中且进一步传递到受体细胞或生物体的后代细胞或者由受体细胞或生物体的后代细胞或生物体遗传。最后,所导入的核酸可仅短暂地存在于受体细胞或者宿主生物体中。
术语“可选择的标记基因”是指当表达时为被转化细胞或植物赋予可选的表现型(诸如抗生素抗性)的基因。
术语“可操作地连接的”表示将对于编码序列的表达所必需的调控序列置于DNA分子中相对于编码序列的适当位置,从而影响编码序列的表达。有时将该相同的定义应用于转录单位和其它转录控制元件(例如增强子)在表达载体中的布置。
本文中使用的术语“寡核苷酸”是指本发明的序列、引物和探针,并且被定义为由两个或两个以上优选地多于三个的核糖核苷酸或脱氧核糖核苷酸所构成的核酸分子。寡核苷酸的精确尺寸将取决于各种因素以及寡核苷酸的具体应用和用途。
词组“特异性地杂交”是指在充分互补序列的两个单链核酸分子之间的缔合,以便在本领域中通常使用的预定条件下实现这种杂交(有时被称为“大体上互补的”)。具体地,该术语是指利用大体互补序列使包含在本发明的单链DNA或RNA分子中的寡核苷酸杂交,以大体上排除寡核苷酸与非互补序列的单链核酸的杂交。
本文中使用的术语“引物”是指单链或双链的DNA寡核苷酸,来源于生物***,由限制性酶内切产生,或者通过合成而产生,当置于适当的环境中时能够功能性地用作模板依赖性核酸合成的引发剂。当提供有适当的核酸模板、合适的核酸的三磷酸核苷前体、聚合酶、合适的辅因子、和条件(诸如合适的温度和pH值)时,引物可以利用聚合酶的作用或者类似的活性通过添加核苷酸在其3'末端延伸以获得引物延伸产物。引物的长度可以基于应用的具体条件和要求而变化。例如,在诊断应用中,寡核苷酸引物的长度通常为15-25个或更多的核苷酸。引物必须具有充分的针对期望模板的互补性,用以启动期望延伸产物的合成,亦即,能够以足以在用于由聚合酶或者类似酶引发的合成的适当的邻近位提供引物的3'羟基基团的方式,与期望的模板链复性。不要求引物序列代表期望的模板的精确互补。例如,非互补的核苷酸序列可连接到互补引物的5'末端。可替代地,非互补碱基可散布在寡核苷酸引物序列中,前提是引物序列与期望的模板链的序列具有充分的互补性,以便功能性地提供用于延伸产物合成的模板-引物复合物。
美国专利第4,683,195、4,800,195、和4,965,188号中已描述了聚合酶链反应(PCR),它们的全部公开内容以参考的方式并入本文中。
术语“分离的”可以是指已充分地与将会自然地结合的其它化合物分离的化合物或复合物。“分离的”并非意图排除与其它化合物或材料的人工或合成混合物,也非意图排除不影响基本活性或者接下来的测定并且会例如由于不完全的纯化或者稳定剂的添加而存在的杂质的存在。
术语“免疫应答”意图是指脊椎动物受试对象的免疫***对抗原或抗原决定簇的任何应答。示例性的免疫应答包括:体液免疫应答(例如抗原特异性抗体的产生)和细胞介导的免疫应答(例如淋巴细胞增殖),如下文中的定义。
II.本发明变异体AAV载体的使用方法和给予方法
本发明的方法提供一种用于将异源核酸序列输送入大范围宿主细胞(包括***和非***细胞)中的手段。另外,作为治疗的方法等,本发明的载体和其它试剂、方法和药物制剂可用于将蛋白质或肽给予有需要的受试对象的方法。因此,可以这种方式在受试对象体内产生蛋白质和肽。作为治疗的方法等,受试对象会需要蛋白质或肽,因为受试对象具有蛋白质或肽的缺乏,或者因为在受试对象中蛋白质或肽的产生会赋予一些治疗性作用,如下面进一步的说明。
一般来说,本发明可用于传输具有生物学效应的任何外来核酸,以便治疗或减轻与有关基因表达的任何疾病相关的症状。说明性的疾病状态包括但不限于:囊性纤维化(和其它肺病)、血友病A、血友病B、珠蛋白生成障碍性贫血、贫血症和其它血液凝固障碍、艾滋病、阿尔茨海默氏病、帕金森氏病、亨廷顿舞蹈病、肌萎缩侧索硬化、癫痫、和其它神经***疾病、癌症、糖尿病、肌肉萎缩症(例如,Duchenne氏、Becker氏)、高雪氏病、赫尔勒氏病、腺苷脱氨酶缺乏症、糖原贮积病和其它代谢缺陷、视网膜退行性疾病(和其它眼病)、实体器官的疾病(例如,大脑、肝脏、肾脏、心脏)等。
另外,本发明可用于传输已知提供有利的生物学效果以便治疗或减轻与癌症、传染病、和诸如类风湿关节炎在内的自身免疫病相关的症状的编码单克隆抗体的核酸或者其片段。其它序列可编码例如细胞因子,诸如可调节疾病过程的α-干扰素。
基因转移在了解和提供疾病状态的治疗中具有显著的潜在用途。存在一些其缺陷基因是已知的并且已被克隆的遗传性疾病。在有些情况下,这些克隆化基因的功能是已知的。一般来说,上述疾病状态分为两种类型:通常以隐形的方式遗传的缺陷状态,通常是酶的缺陷状态;和不平衡状态,其至少有时与以显性方式遗传的调控或结构蛋白有关。就缺乏状态疾病而言,可以利用基因转移使正常的基因进入受感染的组织用于替代治疗,以及利用反义突变建立疾病的动物模型。就不平衡疾病状态而言,基因转移可以用于形成模型***中的疾病状态,然后可以用于致力于对抗疾病状态。因此,本发明的方法能够治疗遗传性疾病。本文中使用的“疾病状态”是通过部分或完全地治疗导致疾病或者使它更加严重的缺乏或不平衡疾病而得以治疗。导致突变或纠正缺陷的核酸序列的位点特异性整合的应用也是可行的。
最后,本发明还可应用于诊断和筛选方法,由此在细胞培养***或者可替代地转基因动物模型中短暂地或稳定地表达所研究的基因。
III.受试对象、药物制剂、疫苗、和给药方式
本发明可用于兽医和医学应用。合适的受试对象包括禽类和哺乳动物,其中优选的是哺乳动物。本文中使用的术语“禽类”包括但不限于:鸡、鸭、鹅、鹌鹑、火鸡和雉鸡。本文中使用的术语“哺乳动物”包括但不限于:人类、牛类、绵羊类、山羊类、马类、猫类、犬类、兔类等。人受试对象是最优选的。人受试对象包括胎儿、新生儿、幼儿、青少年和成人受试对象。
在具体的实施方式中,本发明提供了一种包含在药学上可接受的载体或者其它医用剂、药用剂、载体、佐剂、稀释剂等中的本发明的病毒颗粒的药物组合物。就注射而言,载体通常将是液体。就其它给药方法而言,载体可以是固体或者液体,诸如无菌无热原水或者无菌无热原磷酸盐缓冲生理盐水。就吸入给药而言,载体将是可吸入的,并且将优选地采用固体或液体颗粒形式。作为注射介质,优选的是使用含有可用于注射液的添加剂(诸如稳定剂、盐类或生理盐水、和/或缓冲剂)的水。
在其它实施方式中,本发明提供一种包含在药学上可接受的载体或者其它医用剂、药用剂、载体、佐剂、稀释剂等中的细胞的药物组合物;在该细胞中将AAV原病毒结合入基因组。
“药学上可接受的”表示并非生物学不合适的物质,例如可在不引起任何不良生物学效应的情况下将该物质给予受试对象。因此,这种药物组合物可以用于例如细胞的离体转染,或者用于将病毒颗粒或细胞直接地给予受试对象。
本发明还提供一种将核酸传递至细胞的方法。就体外方法而言,可利用如本领域已知的标准病毒转导方法将病毒提供给细胞。优选地,按照适合于特定靶细胞的标准转导方法,以适当的多重性感染将病毒颗粒加入到细胞中。所给予的病毒滴度可以根据靶细胞类型和具体的病毒载体而变化,并且可以由本领域技术人员在不需要过度实验的情况下而决定。可替代地,本发明细小病毒载体的给予可以利用本领域已知的任何其它方法而完成。
优选地,将重组病毒载体以生物有效量给予细胞。病毒载体的“生物有效”量是足以导致异源核酸序列在细胞中感染(或转导)和表达的量。如果将病毒体内给予细胞(例如,将病毒给予受试对象,如下所述),则病毒载体的“生物有效”量是足以导致在靶细胞中异源核酸序列的转导和表达的量。
被给予本发明病毒载体的细胞可属于任何类型,包括但不限于:神经细胞(包括周围和中枢神经***的细胞,特别是脑细胞)、肺细胞、视网膜细胞、上皮细胞(例如,肠道和呼吸道上皮细胞)、肌肉细胞、胰腺细胞(包括胰岛细胞)、肝细胞、心肌细胞、骨细胞(例如,骨髓干细胞)、造血干细胞、脾脏细胞、角质形成细胞、成纤维细胞、内皮细胞、***细胞、生殖细胞等。可替代地,细胞可以是任何祖细胞。作为另一个替代物,细胞可以是干细胞(例如,神经干细胞、肝干细胞)。此外,细胞可以是来自于任何种类的来源,如上面所指出。
在本发明的具体实施方式中,从受试对象中取出细胞,将细小病毒载体导入其中,然后将细胞替换回受试对象中。从受试对象中取出细胞用于离体治疗接着导入受试对象中方法的在本领域是已知的。可替代地,将rAAV载体导入来自另一个受试对象的细胞,导入培养的细胞中,或者导入来自任何其它合适来源的细胞,并且将细胞给予需要它的受试对象。
用于离体基因治疗的合适细胞包括但不限于:肝细胞、神经细胞(包括中枢神经***和周围神经***的细胞,特别是脑细胞)、胰腺细胞、脾脏细胞、成纤维细胞(例如,皮肤成纤维细胞)、角质形成细胞、内皮细胞、上皮细胞、成肌细胞、造血细胞、骨髓***、祖细胞、和干细胞。
给予受试对象的细胞的剂量将根据受试对象的年龄、主体的病情和种属、细胞的类型、细胞所表达的核酸、给药方式等而变化。通常,每个剂量将给予至少大约102至大约108、优选地大约103至大约106个细胞。优选地,以“治疗有效量”给予细胞。
本文中使用的“治疗有效”量是足以缓解(例如,减轻、减弱、减小)与疾病相关的至少一种症状的量。可替代地,“治疗有效”量是足以提供对受试对象病情的一些改善的量。
本发明的另一方面是一种用本发明的病毒颗粒对受试对象进行体内治疗的方法。本发明的细小病毒颗粒向有需要的人受试对象或动物的给药,可以采用本领域中已知的用于给予病毒载体的任何方法。
示例性的给药方式包括:口服、直肠、经粘膜、局部、经皮、吸入、胃肠外(例如,静脉、皮下、皮内、肌肉、和关节内)给药等方式,以及直接组织或器官注射,可替代地,鞘内、直接肌肉、心室内给药、静脉给药、腹腔内给药、鼻内给药、或眼内注射。可以以常规的方式将注射剂制备成溶液剂或悬浮剂、适合用于注射前的液体中的溶液剂或悬浮剂的固体形式、或者乳剂的形式。可替代地,可以局部而不是***的方式给予例如在贮库或者缓释制剂中的病毒。
在本发明的具体执行的实施方式中,将所研究的核苷酸序列传输至受试对象的肝脏。可利用本领域已知的任何方法实现对肝的给药,包括但不限于:静脉给药、门静脉给药、胆管给药、动脉内给药、和直接注射入肝实质。
优选地,利用编码肽或蛋白质的重组细小病毒载体来感染细胞(例如,肝细胞),这些细胞表达编码的肽或蛋白质并且以治疗有效量将其分泌入循环***(如上所定义的)。可替代地,利用另一种细胞或组织(包括但不限于大脑、胰腺、脾脏或肌肉)来传递和表达载体。
在其它优选实施方式中,将本发明的细小病毒颗粒通过肌肉注射给药,更优选地利用肌肉注射或者利用局部给药(如上所定义的)。在其它优选实施方式中,将本发明的细小病毒颗粒给予肺。
可利用任何合适的方法将本文中公开的细小病毒载体给予受试对象的肺,但优选地通过给予由受试对象所吸入的本发明的细小病毒载体所构成的可吸入颗粒的气雾剂悬浮液的方式而给予。可吸入颗粒可以是液体或者固体。可以利用任何合适的方式(诸如用压力驱动的气雾剂雾化器或者超声雾化器)来制造包含本发明的细小病毒载体的液体颗粒的气雾剂,正如本领域技术人员所了解的。参见例如美国专利No.4,501,729。包含本发明病毒载体的固体颗粒的气雾剂,同样地可利用药学行业中已知的技术用任何固体颗粒药物气雾剂发生器而制造。
本发明的细小病毒颗粒的剂量将基于给药方式、拟被治疗的疾病或病情、单独受试对象的病情、具体的病毒载体、拟被给予的基因;并且可以常规的方式来决定。用于获得治疗效果的示例性剂量为至少大约105、106、107、108、109、1010、1011、1012、1013、1014、1015、1016个或更多转导单元、或者更优选大约108至1013个转导单元、更优选1012个转导单元的病毒滴度。
总之,本发明的细小病毒载体、试剂、和方法可以应用将核酸引导到***的细胞或者非***的细胞,并且稳定地在其中表达异源核酸。利用此载体***,现在可以以体外或体内方式将编码影响细胞生理的蛋白质的基因导入细胞。因此,本发明的载体可以用于疾病状态的基因治疗、或者用于细胞生理的实验性修改。
提供以下的实施例来说明本发明的某些实施方式。其目的不是以任何方式限制本发明。
实施例I
赖氨酸到精氨酸突变影响腺相关病毒转导率和MHC给药
AAV1和AAV8载体中被靶向的赖氨酸残基的鉴定
我们利用UbPred软件来预测在AAV1、AAV2、AAV8和Rh74衣壳蛋白上的可能的泛素化位点(Radivojac P et al,2010)。UbPred软件从网站http://www.ubpred.org/ index.html在线获得。分析的输出是对在指定的腺相关病毒血清型衣壳序列中的泛素化而言重要的赖氨酸残基的预测。见图1和表1。这些如下:
AAV1衣壳蛋白VP1序列:
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLKAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEQSPQEPDSSSGIGKTGQQPAKKRLNFGQTGDSESVPDPQPLGEPPATPAAVGPTTMASGGGAPMADNNEGADGVGNASGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISSASTGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTTNDGVTTIANNLTSTVQVFSDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEEVPFHSSYAHSQSLDRLMNPLIDQYLYYLNRTQNQSGSAQNKDLLFSRGSPAGMSVQPKNWLPGPCYRQQRVSKTKTDNNNSNFTWTGASKYNLNGRESIINPGTAMASHKDDEDKFFPMSGVMIFGKESAGASNTALDNVMITDEEEIKATNPVATERFGTVAVNFQSSSTDPATGDVHAMGALPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKNPPPQILIKNTPVPANPPAEFSATKFASFITQYSTGQVSVEIEWELQKENSKRWNPEVQYTSNYAKSANVDFTVDNNGLYTEPRPIGTRYLTRP
赖氨酸
图例:
AAV-1中的期望的突变体
ID | AAV-1突变体 |
MUT1-1 | K61R |
MUT1-2 | K84R |
MUT1-3 | K137R |
MUT1-4 | K143R |
MUT1-5 | K161R |
MUT1-6 | K459R |
MUT1-7 | K528R |
MUT1-8 | K533R |
MUT1-9 | K707R |
AAV8衣壳蛋白VP1序列:
MAADGYLPDWLEDNLSEGIREWWALKPGAPKPKANQQKQDDGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVEEGAKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPARKRLNFGQTGDSESVPDPQPLGEPPAAPSGVGPNTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGATNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLSFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFQFTYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQTTGGTANTQTLGFSQGGPNTMANQAKNWLPGPCYRQQRVSTTTGQNNNSNFAWTAGTKYHLNGRNSLANPGIAMATHKDDEERFFPSNGILIFGKQNAARDNADYSDVMLTSEEEIKTTNPVATEEYGIVADNLQQQNTAPQIGTVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQSKLNSFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTSVDFAVNTEGVYSEPRPIGTRYLTRNL
赖氨酸
图例:
所附的表3提供利用不同衣壳突变体所获得的载体的产量,包括本发明的AAV8。该表也显示当与相同血清型的野生型载体相比较时突变是否导致提高的、降低的或相当的转导效率。
AAV2衣壳蛋白VP1序列:
MAADGYLPDWLEDTLSEGIRQWWKLKPGPPPPKPAERHKDDSRGLVLPGYKYLGPFNGLDKGEPVNEADAAALEHDKAYDRQLDSGDNPYLKYNHADAEFQERLKEDTSFGGNLGRAVFQAKKRVLEPLGLVEEPVKTAPGKKRPVEHSPVEPDSSSGTGKAGQQPARKRLNFGQTGDADSVPDPQPLGQPPAAPSGLGTNTMATGSGAPMADNNEGADGVGNSSGNWHCDSTWMGDRVITTSTRTWALPTYNNHLYKQISSQSGASNDNHYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNDGTTTIANNLTSTVQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMVPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFTFSYTFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTNTPSGTTTQSRLQFSQAGASDIRDQSRNWLPGPCYRQQRVSKTSADNNNSEYSWTGATKYHLNGRDSLVNPGPAMASHKDDEEKFFPQSGVLIFGKQGSEKTNVDIEKVMITDEEEIRTTNPVATEQYGSVSTNLQRGNRQAATADVNTQGVLPGMVWQDRDVYLQGPIWAKIPHTDGHFHPSPLMGGFGLKHPPPQILIKNTPVPANPSTTFSAAKFASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYNKSVNVDFTVDTNGVYSEPRPIGTRYLTRNL
赖氨酸
图例:
AAV-Rh74衣壳蛋白VP1序列
MAADGYLPDWLEDNLSEGIREWWDLKPGAPKPKANQQKQDNGRGLVLPGYKYLGPFNGLDKGEPVNAADAAALEHDKAYDQQLQAGDNPYLRYNHADAEFQERLQEDTSFGGNLGRAVFQAKKRVLEPLGLVESPVKTAPGKKRPVEPSPQRSPDSSTGIGKKGQQPAKKRLNFGQTGDSESVPDPQPIGEPPAGPSGLGSGTMAAGGGAPMADNNEGADGVGSSSGNWHCDSTWLGDRVITTSTRTWALPTYNNHLYKQISNGTSGGSTNDNTYFGYSTPWGYFDFNRFHCHFSPRDWQRLINNNWGFRPKRLNFKLFNIQVKEVTQNEGTKTIANNLTSTIQVFTDSEYQLPYVLGSAHQGCLPPFPADVFMIPQYGYLTLNNGSQAVGRSSFYCLEYFPSQMLRTGNNFEFSYNFEDVPFHSSYAHSQSLDRLMNPLIDQYLYYLSRTQSTGGTAGTQQLLFSQAGPNNMSAQAKNWLPGPCYRQQRVSTTLSQNNNSNFAWTGATKYHLNGRDSLVNPGVAMATHKDDEERFFPSSGVLMFGKQGAGKDNVDYSSVMLTSEEEIKTTNPVATEQYGVVADNLQQQNAAPIVGAVNSQGALPGMVWQNRDVYLQGPIWAKIPHTDGNFHPSPLMGGFGLKHPPPQILIKNTPVPADPPTTFNQAKLASFITQYSTGQVSVEIEWELQKENSKRWNPEIQYTSNYYKSTNVDFAVNTEGTYSEPRPIGTRYLTRNL
赖氨酸
图例:
AAV-rh74中的理想的突变体(也见表3)
ID | AAV-rh74突变体 |
Mut74-1 | K26R |
Mut74-2 | K31R |
Mut74-3 | K33R |
Mut74-4 | K38R |
Mut74-5 | K51R |
Mut74-6 | K77R |
Mut74-7 | K137R |
Mut74-8 | K163R |
Mut74-9 | K169R |
Mut74-10 | K259R |
Mut74-11 | K333R |
Mut74-12 | K530R |
Mut74-13 | K547R |
Mut74-14 | K552R |
Mut74-15 | K569R |
Mut74-16 | K668R |
Mut74-17 | K691R |
Mut74-18 | K695R |
Mut74-19 | K709R |
利用下面的引物组形成本发明的含赖氨酸的衣壳变异体:
用于诱变的引物:
·AAV1引物:
AAV1 K61R
正义:5'-CTT CAA CGG ACT CGA CAG GGG GGA GCC-3'
反义:5'-GGC TCC CCC CTG TCG AGT CCG TTG AAG-3'
AAV1 K84R
正义:5'-GCC TAC GAC CAG CAG CTC AGA GCG GGT GAC-3'
反义:5'-GTC ACC CGC TCT GAG CTG CTG GTC GTA GGC-3'
AAV1 K137R
正义:5'-TGG TTG AGG AAG GCG CTA GGA CGG CTC CT-3'
反义:5'-AGG AGC CGT CCT AGC GCC TTC CTC AAC CA-3'
AAV1 K143R
正义:5'-CTA AGA CGG CTC CTG GAA AGA GAC GTC CGG TAG-3'
反义:5'-CTA CCG GAC GTC TCT TTC CAG GAG CCG TCT TAG-3'
AAV1 K161R
正义:5'-CGG GCA TCG GCA GGA CAG GCC AGC A-3'
反义:5'-TGC TGG CCT GTC CTG CCG ATG CCC G-3'
AAV1 K459R
正义:5'-AGT CCG GAA GTG CCC AAA ACA GGG ACT TGC TGT-3'
反义:5'-ACA GCA AGT CCC TGT TTT GGG CAC TTC CGG ACT-3'
AAV1 K528R
正义:5'-GCA CTG CTA TGG CCT CAC ACA GAG ACG ACG AAG-3'
反义:5'-CTT CGT CGT CTC TGT GTG AGG CCA TAG CAG TGC-3'
AAV1 K533R
正义:5'-CAA AGA CGA CGA AGA CAG GTT CTT TCC CAT GAG CG-3'
反义:5'-CGC TCA TGG GAA AGA ACC TGT CTT CGT CGT CTT TG-3'
AAV1 K707R
正义:5'-TGCAGTACACATCCAATTATGCAAGATCTGCCAACGTTG-3'
反义:5'-CAACGTTGGCAGATCTTGCATAATTGGATGTGTACTGCA-3'
AAV8引物
AAV8 K137R
正义:5'-GGT TGA GGA AGG CGC TAG GAC GGC TCC TGG-3'
反义:5'-CCA GGA GCC GTC CTA GCG CCT TCC TCA ACC-3'
AAV8 K333R
正义:5'-GCA GAA TGA AGG CAC CAG GAC CAT CGC CAA TAA CC-3'
反义:5'-GGT TAT TGG CGA TGG TCC TGG TGC CTT CAT TCT GC-3'
AAV8 K530R
正义:5'-GCA TCG CTA TGG CAA CAC ACA GAG ACG ACG AGG-3'
反义:5'-CCT CGT CGT CTC TGT GTG TTG CCA TAG CGA TGC-3'
AAV8 K709R
正义:5’-GTACACCTCCAACTACTACAGATCTACAAGTGTGGACTTTG-3'
反义:5'-CAAAGTCCACACTTGTAGATCTGTAGTAGTTGGAGGTGTAC-3'
AAV2引物
AAV2 K39R
正义:5'-GCCCGCAGAGCGGCATAGGGACGACAG-3'
反义:5’-CTGTCGTCCCTATGCCGCTCTGCGGGC-3’
AAV2 K137R
正义:5'-CCTGGTTGAGGAACCTGTTAGGACGGCTCCGG-3'
反义:5’-CCGGAGCCGTCCTAACAGGTTCCTCAACCAGG-3’
AAV2 K143R
正义:5'-AGACGGCTCCGGGAAAAAGGAGGCCGGTA-3'
反义:5'-TACCGGCCTCCTTTTTCCCGGAGCCGTCT-3'
AAV2 K161R
正义:5'-CCTCGGGAACCGGAAGGGCGGGCC-3'
反义:5'-GGCCCGCCCTTCCGGTTCCCGAGG-3'
AAV2 K490R
正义:5'-CCGCCAGCAGCGAGTATCAAGGACATCTGCGG-3'
反义:5'-CCGCAGATGTCCTTGATACTCGCTGCTGGCGG-3'
AAV2 K527R
正义:5'-CGGCCATGGCAAGCCACAGGGACGATGAA-3'
反义:5'-TTCATCGTCCCTGTGGCTTGCCATGGCCG-3'
AAV2 K532R
正义:5'-ACAAGGACGATGAAGAAAGGTTTTTTCCTCAGAGCGG-3'
反义:5'-CCGCTCTGAGGAAAAAACCTTTCTTCATCGTCCTTGT-3'
AAV-rh74引物
AAV-rh74 K137R
正义:5'-CTGGTTGAATCGCCGGTTAGGACGGCTCCTG-3'
反义:5'-GACCAACTTAGCGGCCAATCCTGCCGAGGAC-3'
AAV-rh74 K333R
正义:5'-GCAGAATGAAGGCACCAGGACCATCGCCAATAACC-3'
反义:5'-GGTTATTGGCGATGGTCCTGGTGCCTTCATTCTGC-3'
AAV-rh74 K530R
正义:5'-GTTGCCATGGCTACCCACAGGGACGACGAA-3'
反义:5'-TTCGTCGTCCCTGTGGGTAGCCATGGCAAC-3'
AAV-rh74 K552R
正义:5'-GGAAACAGGGAGCTGGAAGAGACAACGTGGACTAT-3'
反义:5'-ATAGTCCACGTTGTCTCTTCCAGCTCCCTGTTTCC-3'
AAV-rh74 K569R
正义:5'-CTAACCAGCGAGGAAGAAATAAGGACCACCAACCC-3'
反义:5'-GGGTTGGTGGTCCTTATTTCTTCCTCGCTGGTTAG-3'
AAV-rh74 K691R
正义:5'-CGAGTGGGAGCTGCAGAGGGAGAACAGCAA-3'
反义:5'-TTGCTGTTCTCCCTCTGCAGCTCCCACTCG-3'
AAV-rh74 K695R
正义:5'-GCTGCAGAAGGAGAACAGCAGACGCTGGAACC-3'
反义:5'-GGTTCCAGCGTCTGCTGTTCTCCTTCTGCAGC-3'
AAV-rh74 K709R
正义:5'-AGTACACTTCCAACTACTACAGATCTACAAATGTGGACTTTGC-3'
反义:5'-GCAAAGTCCACATTTGTAGATCTGTAGTAGTTGGAAGTGTACT-3'
下表提供通过用于包装FIX的肝特异性AAV转基因表达盒的诱变所获得的一系列的AAV载体。基于野生型未修饰AAV8载体中所观察结果难区分包装效率。
在本文中所描述的任何衣壳蛋白中含有2、3、4、5、6、7或更多的改变的赖氨酸残基以便进一步提高转导效率的衣壳突变体,也是在本发明的范围内。数据也表明某些突变形成显示显著降低的转导效率的变异体。这种变异体可以与显示提高的转导效率的变异体结合使用,从而用作使抗体引导的针对进入的载体的免疫应答中和或饱和的诱饵,由此使载体能够携带期望的转基因以便更高效率地进入细胞。
图1示出了衣壳表面的示意图。图2A、图2B和图2C中所给出的数据表明改变AAV8上的VP1衣壳中的赖氨酸残基由于改变的转导水平可改变所产生的转基因的水平。图3A、图3B和图3C示出了单个和多个突变对转导细胞中的HF.IX产生的影响。图3D显示例如3个赖氨酸向精氨酸残基与4或6个酪氨酸向苯丙氨酸残基的组合突变降低转导率。
利用AAV赖氨酸突变体对HHL5-B7肝细胞的CTL杀伤
我们对用这里所公开的某些AAV赖氨酸突变体而转导的肝细胞的CTL杀伤进行了评估。利用以下的材料和方法来评估转导的肝细胞的CTL杀伤。
载体产生
利用如前所述的三重转染法(Matsushita,1998年)在HEK-293细胞中制造AAV载体,利用绿化铯梯度离心法(Ayuso,2010年)进行纯化。利用Genemed合成法合成出AAV表位肽,将其以5mg/ml的浓度再悬浮于100%DMSO中。
T细胞的体外扩增
将人PBMC(Cellular Technology有限公司)解冻、清洗、计数,以2×106个细胞/ml的浓度再悬浮于含有3%人血清(Bioreclamation)、1%L-谷氨酰胺(Gibco)、和1%的青霉素/链霉素(Gibco)的AIM-V淋巴细胞培养基(Gibco)中。对于各扩增条件而言,以500μl的体积将1×106(500μl)个细胞/孔添加在24孔板(BD Falcon)中。也将另外的1×106(500μl)的自体放射线照射脾细胞(3000rad)添加到各孔中作为饲养细胞,以及2.5μg/ml的人β-2-微球蛋白(Lee Biosolutions)、和10ng/ml的人重组IL-7(R&D Systems)。使细胞在AAV肽的存在下以10μg/ml的最终浓度在37℃5%CO2条件下扩大。在前24小时后,将人IL-2(Roche)以10ng/ml的浓度添加到细胞培养物中,其后每48小时补充一次。必要时,将细胞划分入新的孔中,每7-10天重复抗原刺激(抗原和饲养细胞)多达3轮的重新刺激。
CTL测定
以如前所述的方式执行CTL测定(Pien,2009)。简略地,用CytoTox 96非放射性细胞毒性检测(Promega)测量在CTL介导的靶细胞溶解之后的乳酸脱氢酶(LDH)。将4千个HHL5肝细胞靶细胞覆盖在Microtest Primaria平底96孔板(BD Falcon)的各孔中的不含血清的DMEM中。以5000、50,000、和500,000MOI的AAV衣壳对靶细胞进行转导,在37℃ 5%CO2条件下保温培养18小时。在处理和保温培养之后,用培养基将覆盖的靶细胞清洗一次,然后添加表位特异性细胞毒T淋巴细胞,以如上所述的方式扩大。在37℃、5%CO2条件下以10:1的效应子/靶细胞比率添加CTL达4小时,在室温下进行30分钟保温培养后用在490nm处利用分光光度计(Spectramax)的酶底物读数来测量LDH。
流式细胞仪
在AAV转导之后利用流式细胞仪测量GFP表达。将取自细胞系HHL5或Huh7的人肝细胞以250,000细胞/孔的密度覆盖在Primaria Multiwell 24孔板中的(BD Falcon)含有10%胎牛血清、1%L-谷氨酰胺(Gibco)、和1%青霉素/链霉素(Gibco)的DMEM中。用5000、50000或5000000MOI的AAV载体对细胞进行转导,并且于37℃、5%CO2中保温培养18小时。在保温培养后,对细胞进行胰蛋白酶处理,用PBS 2%FBS清洗二次,用2%多聚甲醛固定。在使用(BD Biosciences)的FACS Canto II流式细胞仪中获取样品,利用软件(Treestar)进行进一步的分析。
CTL测定结果
为了进一步测试赖氨酸突变对病毒转导的影响,我们利用之前由我们实验室所开发的体内CTL介导的细胞毒性检测来测试AAV载体的功能(Pien et al)。将AAV1和AAV2转导结果示于图4和图5和图6。在图4中,AAV-1衣壳中的所有赖氨酸突变导致靶细胞的CTL介导杀伤的减小,从而表明赖氨酸突变导致转导时效率降低的表面抗原的处理和呈现。此外,赖氨酸突变的影响似乎是相加的,其中三倍和四倍赖氨酸突变体显示最大的CTL介导杀伤的减小(图4A、图4B)。向AAV-2衣壳的突变显示类似的作用。见图5和图6。图7示出了当对Rh74变异体进行测试时所获得的转导结果。
总之,我们已发现使AAV衣壳上的赖氨酸残基向精氨酸残基突变提高了AAV转导效率。我们的实验鉴定出若干个变异体,这些变异体在转导时导致与接受未修饰AAV载体相比在小鼠与动物中人第九因子(FIX)转基因的较高水平的表达。
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虽然上面已描述并具体例示了本发明的某些优选实施方式,但并非意图是本发明局限于上述实施方式。可在不背离如所附权利要求中所陈述本发明范围和精神的前体下,对这些实施方式作出种各种修改。
表1
AAV1、AAV2和AAV8赖氨酸位置以及它们的泛素化
颜色标记:绿色:低置信度,蓝色:中置信度,红色:高置信度,黑色:无泛素化预测
表2
AAV8衣壳的泛素化预测
赖氨酸 | 表面 | 经过ubipred的预测 | 所产生的载体 |
26 | NA(不在VP3) | ||
31 | NA(不在VP3) | ||
33 | NA(不在VP3) | ||
38 | NA(不在VP3) | 低置信度 | 是 |
51 | NA(不在VP3) | ||
61 | NA(不在VP3) | 低置信度 | |
77 | NA(不在VP3) | 低置信度 | |
122 | NA(不在VP3) | ||
123 | NA(不在VP3) | ||
137 | NA(不在VP3) | 高置信度 | 是 |
142 | NA(不在VP3) | ||
143 | NA(不在VP3) | 低置信度 | 是 |
162 | NA(不在VP3) | ||
163 | NA(不在VP3) | ||
170 | NA(不在VP3) | ||
259 | 是 | 是 | |
312 | |||
317 | |||
324 | |||
333 | 是 | 中置信度 | 是 |
478 | |||
510 | 是 | 是 | |
530 | 中置信度 | 是 | |
547 | 是 | 是 | |
569 | 是 | 低置信度 | 是 |
623 | |||
643 | |||
652 | 未包装的 | ||
668 | 是 | 低置信度 | 是 |
691 | 低置信度 | ||
695 | 低置信度 | ||
709 | 是 | 低置信度 | 是 |
UbPred软件(http://www.ubpred.org/):
Radivojac P.,Vacic,V.,Haynes,C.,Cocklin,R.R.,Mohan,A.,Heyen,J.W.,Goebl,M.G.,and Iakoucheva,L.M.Identification,Analysis and Prediction ofProtein Ubiquitination Sites.Proteins:Structure,Function,andBioinformatics.78(2):365-380.(2010)
表3
MHC传送
定义TMR=K(137/333/530)R
定义HMF=Y(253/275/447/703/707/733)F
定义HMR:K(137/259/333/530/552/569)R
定义195/202:G195A+L199V+S201P+G202N
Claims (24)
1.一种腺相关病毒(AAV)载体,所述载体包含VP1衣壳蛋白,所述VP1衣壳蛋白包含如下一个或多个赖氨酸替换:在AAV1 VP1衣壳蛋白的位点137,K137R;位点459,K459R;位点528,K528R;位点533,K533R;位点707,K707R或位点K137R/K459R/K533R,所述载体还包含小基因,所述小基因包含AAV反向末端重复和能操作连接到调控序列的异源核酸序列,所述调控序列引导在宿主细胞中由所述异源核酸序列对产物的表达,所述赖氨酸替换对于抑制所述衣壳蛋白的泛素化是有效的,由此相较于所包含的AAV1VP1衣壳蛋白不具有一个或多个赖氨酸替换的AAV载体增加所述AAV载体向靶细胞中的转导。
2.一种腺相关病毒(AAV)载体,所述载体包含VP1衣壳蛋白,所述VP1衣壳蛋白包含如下一个或多个赖氨酸替换:在AAV2VP1衣壳蛋白的位点137,K137R;位点527,K527R;或位点532,K532R,所述载体还包含小基因,所述小基因包含AAV反向末端重复和能操作连接到调控序列的异源核酸序列,所述调控序列引导在宿主细胞中由所述异源核酸序列对产物的表达,所述赖氨酸替换对于抑制所述衣壳蛋白的泛素化是有效的,由此相较于所包含的AAV2VP1衣壳蛋白不具有一个或多个赖氨酸替换的AAV载体增加所述AAV载体向靶细胞中的转导。
3.一种腺相关病毒(AAV)载体,所述载体包含VP1衣壳蛋白,所述衣壳蛋白具有在AAV8VP1衣壳蛋白的位点569,K569R,位点668,K668R或K137R/K333R/K530R中的一或多个赖氨酸替换,所述载体还包含小基因,所述小基因包含AAV反向末端重复和能操作连接到调控序列的异源核酸序列,所述调控序列引导在宿主细胞中由所述异源核酸序列对产物的表达,所述赖氨酸替换对于抑制所述衣壳蛋白的泛素化是有效的,由此相较于所包含的AAV8VP1衣壳蛋白不具有一个或多个赖氨酸替换的AAV载体增加所述AAV载体向靶细胞中的转导。
4.一种腺相关病毒(AAV)载体,所述载体包含VP1衣壳蛋白,所述衣壳蛋白具有AAV-rh74VP1衣壳蛋白的K137/333/530/552R替换;K137/259/333/530/552/569R+G195A+L199V+S201P+G202N替换;K137/259/333/530/552/569R+G195A+L199V+S201P+G202N+K38R替换;K137/259/333/530/552/569R+G195A+L199V+S201P+G202N+K51R替换;K137/259/333/530/552/569R+G195A+L199V+S201P+G202N+K61R替换或K137/259/333/530/552/569R+G195A+L199V+S201P+G202N+K77R替换,所述载体还包含小基因,所述小基因包含AAV反向末端重复和能操作连接到调控序列的异源核酸序列,所述调控序列引导在宿主细胞中由所述异源核酸序列对产物的表达,其中赖氨酸替换对于抑制所述衣壳蛋白的泛素化是有效的,由此相较于所包含的AAV-rh74VP1衣壳蛋白不具有一个或多个赖氨酸替换的AAV载体增加所述AAV载体向靶细胞中的转导。
5.如权利要求1-4中任一项所述的AAV载体,其中所述异源核酸序列的表达产物是治疗性肽或核酸。
6.如权利要求5所述的AAV载体,其中所述治疗性肽是选自第八因子、第九因子或者其功能性片段中的凝血因子。
7.如权利要求1-4中任一项所述的AAV载体,其中所述异源核酸序列的表达产物是IgG、IgM、IgA、IgD、IgE、嵌合免疫球蛋白、人源化抗体、或者单链抗体。
8.如权利要求7所述的AAV载体,其中所述异源核酸序列的表达产物是嵌合免疫球蛋白。
9.如权利要求1-4中任一项所述的AAV载体,其中所述异源核酸序列的表达产物是单链抗体。
10.如权利要求1-4中任一项所述的AAV载体,其中所述表达产物是抗病毒RNAi。
11.如权利要求10所述的AAV载体,其中所述RNAi对于抑制HCV的感染和复制是有效的。
12.如权利要求10所述的AAV载体,其中所述RNAi对于抑制真核靶基因的表达是有效的。
13.如权利要求1-4中任一项所述的AAV载体,其中所述异源核酸序列的表达产物是疾病修饰细胞因子。
14.如权利要求4所述的AAV载体,其中所述异源核酸序列的表达产物是成对的锌指核酸酶。
15.如权利要求1-4中任一项所述的AAV载体,所述载体包含2、3或4个赖氨酸替换。
16.一种细胞培养物,包含如权利要求1-4中任一项所述的AAV载体。
17.一种药物组合物,包含如权利要求1-4中任一项所述的AAV载体及其生理学相容的载体。
18.如权利要求17所述的药物组合物,其用于治疗血液、中枢神经***、代谢、肌肉或眼的疾病或紊乱、感染性疾病或遗传性疾病。
19.如权利要求18所述的药物组合物,其中所述异源核酸序列的表达产物是第九因子。
20.如权利要求18所述的药物组合物,其中所述异源核酸序列的表达产物是第八因子。
21.权利要求1-4中任一项所述的AAV载体及其生理学相容的载体在制备用于将异源核酸序列传递至受试对象的细胞的药剂中的用途。
22.如权利要求21所述的用途,所述药剂用于治疗血液、中枢神经***、代谢、肌肉或眼的疾病或紊乱、感染性疾病或遗传性疾病。
23.如权利要求22所述的用途,其中所述异源核酸序列的表达产物是第九因子。
24.如权利要求22所述的用途,其中所述异源核酸序列的表达产物是第八因子。
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