CN115197967A - 制备重组腺相关病毒的辅助质粒及其应用 - Google Patents
制备重组腺相关病毒的辅助质粒及其应用 Download PDFInfo
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Abstract
本公开提供了一种用于制备重组腺相关病毒的辅助质粒,其包括:1)AAV的Rep和Cap蛋白编码序列;2)至少一个启动子序列;以及3)至少一个DA’序列或DA’序列的反向互补序列AD’序列。本公开还提供了该辅助质粒在提高重组腺相关病毒生产能力方面的应用。在加入AD’或DA’序列的情况下,本公开提供的辅助质粒可显著提升细胞的AAV产量。
Description
技术领域
本发明涉及基因工程和在人源细胞中的AAV生产,具体涉及一类新型的AAV包装辅助质粒,及其在AAV生产中的应用。
背景技术
目前,基因治疗从四十年前的科学家的梦想正逐步变成了现实。多种病毒、非病毒载体经过多年的演变、淘汰,目前重组腺相关病毒(rAAV)载体是公认为符合安全性、有效性、特异性三个标准的基因治疗载体。但是,由于业界大规模生产AAV的能力有限,因而临床应用的普及程度也受此制约。
因此,AAV的产量瓶颈亟待打破。在生产上游改进、创新,提高AAV产率是目前降低目前基因治疗生产成本的关键点之一。目前,三质粒转染法简单、快速,是被广泛采用的AAV生产方法。该方法涉及使用三个质粒共转染HEK293细胞:一个辅助质粒pADhelper,提供四个腺病毒元件;一个辅助质粒pRC,提供AAV的Rep和Cap蛋白编码序列,其中Rep蛋白负责AAV基因组的复制与协助AAV基因组颗粒的组装,Cap蛋白组成AAV外壳;一个包括目的序列的质粒,可简称为pGOI,目的序列的上下游各有一个天然AAV基因组中的5’ITR和3’ITR序列。
通常情况下,一个AAV血清型X的pRC质粒中编码序列由来自AAV2的Rep2和编码AAVX的外壳蛋白CapX组成,P5启动子是启动Rep、Cap表达的关键启动子,通常P5启动子置于RepCap编码前,如图2中“P5+RC”所示。X.Xiao等人曾报道在RC的上下游各放置一个P5启动子,可以提高AAV的产率[1]。在目的质粒pGOI中,置于目的基因(或者目的序列)的上下游的ITR,其单个序列全长145bp,被认为与AAV基因组的复制与引导包装相关。因为ITR中存在反向自互补序列,因此在单链DNA的状况下会自互补形成T形的双链结构。T.R.Flotte等人曾发现完整的ITR具有启动子活性,驱动CFTR基因的表达[2]。但是,就驱动基因表达而言,ITR与传统启动子相比,仅具有非常低的转录活性[3]。
发明内容
一方面,本文提供了一种用于制备重组腺相关病毒的辅助质粒,其包括:1)AAV的Rep和Cap蛋白编码序列;2)至少一个启动子序列;以及3)至少一个DA’序列或DA’序列的反向互补序列AD’序列。
在一些实施方案中,所述至少一个DA’或AD’序列位于Rep和Cap蛋白编码序列的上游。
在一些实施方案中,所述至少一个DA’或AD’序列位于所述Rep和Cap蛋白编码序列的下游。
在一些实施方案中,所述辅助质粒从5’至3’方向依次包括:
i)DA’或AD’序列、启动子序列以及Rep和Cap蛋白编码序列;
ii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列以及DA’或AD’序列;
iii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列、DA’或AD’以及启动子序列;或
iv)Rep和Cap蛋白编码序列、DA’或AD’序列以及启动子序列。
在一些实施方案中,所述启动子为P5启动子。
在一些实施方案中,所述P5启动子序列包括SEQ ID NO:2所示序列或与SEQ IDNO:2所示序列具有至少90%序列一致性的功能性变体。
在一些实施方案中,所述DA’序列包括SEQ ID NO:4所示序列或为与SEQ ID NO:4所示序列具有至少90%序列一致性的功能性变体。
在一些实施方案中,所述AD’序列包括SEQ ID NO:3所示序列或为与SEQ ID NO:3所示序列具有至少90%序列一致性的功能性变体。
在一些实施方案中,所述Rep和Cap蛋白来自2型腺相关病毒。
另一方面,本文提供了一种提高重组腺相关病毒生产能力的方法,其包括使用上述辅助质粒转化宿主细胞。
本发明提出一类编码Rep/Cap(包括其所有血清型)辅助包装质粒,在加入AD’或DA’序列的情况下,可显著提升单细胞的AAV产量。
附图说明
图1为AAV基因组ITR结构示意图。
图2为实施例中研究DA’和AD’功能的实验设计示意图。
图3显示了实施例中检测的各组质粒的裂解液病毒滴度与对照载体的比较结果。
具体实施方式
除非另有说明,本文使用的所有技术和科学术语具有本领域普通技术人员所通常理解的含义。
腺相关病毒(adeno-associated virus,AAV):腺相关病毒最早在腺病毒(AdV)制剂中被发现,之后也在人体组织中找到。其属于微小病毒科(Parvoviridae)依赖病毒属(Dependoparvovirus),为结构简单的缺陷型病毒,复制需要辅助病毒(例如腺病毒)的参与。目前认为腺相关病毒不会导致任何人类疾病,这也是将其改造而用于基因治疗的基础。腺相关病毒的基因组大概为4.7kb,为单链DNA分子,包括两个阅读框(rep基因和cap基因)和位于基因组末端的两个反向末端重复(inverted terminal repeat,ITR)。rep基因编码用于病毒复制的多个蛋白(Rep78、Rep68、Rep52和Rep40),cap基因则编码衣壳蛋白的三个亚基(VP1、VP2和VP3)。ITR在腺相关病毒的复制和包装过程中起着关键作用,并且参与了病毒基因组在宿主基因组上的整合和逃逸过程。ITR序列(通常145bp)可形成T型结构。如图1所示,基于在T型结构上的位置,可将ITR序列划分出A、B、B’、C’、C、A’、D和D等多个区段。在序列上A和A’互补,B和B’互补,C和C’互补,D和D’互补。从结构上看,ITR序列包括两个臂回文(B-B’和C-C’)和一个长茎回文(A-A’)。D’区存在AAV基因组5’端,D区段存在于在3’端。
重组腺相关病毒(rAAV):指对野生型腺相关病毒的基因组进行了改进而获得的腺相关病毒。其通常具有野生型AAV的衣壳蛋白和结构,但基因组中的蛋白编码序列(rep和cap基因)被目的基因(例如治疗性基因)所替换,仅保留了两端的ITR序列。rAAV的一个重要用途是作为载体用于基因治疗。
辅助质粒:为用于制备rAAV的辅助质粒。为了产生rAAV,转染宿主细胞(如哺乳动物或昆虫细胞)时通常需要共转染三个质粒:辅助质粒pADhelper,提供四个腺病毒元件;辅助质粒pRC,提供AAV的Rep和Cap蛋白编码序列,其中Rep蛋白负责AAV基因组的复制与协助AAV基因组颗粒的组装,Cap蛋白形成AAV外壳;以及包括目的序列(例如治疗基因)的质粒,可简称为pGOI。
AD’序列:指上文所述A区段和D’区段组合形成的序列。在一个具体实例中,AD’序列具有SEQ ID NO:3所示序列。
DA’序列:指上文所述D区段和A’区段组合形成的序列。在一个具体实例中,DA’序列具有SEQ ID NO:4所示序列。
在本文描述的实施方案中,除非另有说明,AD’序列和DA’序列可互换使用,例如当提及AD’序列时,应理解为也同时提及DA’序列。
序列一致性:提及核苷酸序列时,术语“序列一致性(Sequence identity)”(也称为”序列同一性”)指两核苷酸序列(例如查询序列和参照序列)之间一致性程度的量,一般以百分比表示。通常,在计算两核苷酸序列之间的一致性百分比之前,先进行序列比对(alignment)并引入缺口(gap)(如果有的话)。如果在某个比对位置,两序列中的碱基相同,则认为两序列在该位置一致或匹配;两序列中的碱基不同,则认为在该位置不一致或错配。在一些算法中,用匹配位置数除以比对窗口中的位置总数以获得序列一致性。在另一些算法中,还将缺口数量和/或缺口长度考虑在内。出于本发明的目的,可以采用公开的比对软件BLAST(可在网页ncbi.nlm.nih.gov找到),通过使用缺省设置来获得最佳序列比对并计算出两核苷酸序列之间的序列一致性。
功能性变体:指与作为比较基础的核酸分子相比,在序列上具有至少一个核苷酸变化(包括替换、增加或删除),但这种变化后的核酸分子基本上保留了其原来的生物学功能。在给出示例性核酸分子序列后,本领域技术人员能够通过常规实验方法寻求获得这些功能性变体。因此,一些实施方案中,本文提供的pRC辅助质粒包括的P5启动子序列可包括SEQ ID NO:2所示序列。在另一些实施方案中,该P5启动子序列可为与SEQ ID NO:2所示序列具有至少90%(例如至少95%或至少98%)序列一致性的功能性变体。在一些实施方案中,本文提供的pRC辅助质粒包括的DA’序列可包括SEQ ID NO:4所示序列。在另一些实施方案中,该DA’序列为与SEQ ID NO:4所示序列具有至少90%(例如至少92%、至少95%或至少98%)序列一致性的功能性变体。在一些实施方案中,本文提供的pRC辅助质粒包括的AD’序列可包括SEQ ID NO:3所示序列。在另一些实施方案中,该AD’序列为与SEQ ID NO:3所示序列具有至少90%(例如至少92%、至少95%或至少98%)序列一致性的功能性变体
本发明中涉及的全部pRC系列载体均通过常规的分子克隆方法获得。
在本文提供的pRC辅助质粒中,通过P5启动子和DA’或AD’序列的联合使用,可显著提供rAAV的产量。
以下通过具体实施例来进一步阐述本发明。
实施例
通过设计含有多种DA’序列或AD’和P5启动子的数量和放置位置的组合,通过常规的分子克隆方法基于骨架载体(SEQ ID NO:1)构建出不同的Rep2Cap2候选辅助载体(图2),再平行比较各辅助载体在通用的三质粒转染体系中的产毒滴度。
三质粒转染生产AAV方法简要步骤:
1)铺293T细胞(293T,来源于CRL-3216TM)约3x105至24孔板中,使用高糖DMEM培养基,含10%胎牛血清、2mM L-glutamine(ATCC 30-2214)、1%Penicillin/Streptomycin,在37℃,5%CO2条件下培养约16小时,转染时细胞密度约为60~70%;
2)把pRC与pAdHleper辅助载体(载体来源:美国宾夕法尼亚大学载体部UPENNvector core PL-F-PVADF6)及CAG启动子驱动的荧光蛋白基因的AAV质粒pAAV.CAG.EGFP载体按0.5μg:0.5μg:0.5μg加入0.5mL DMEM中,再加入PEI 3μL(1μg/μL),马上混匀,常温下放置10分钟后,吸去24孔板中培养基,加入转染混合培养基中再放回37℃细胞培养箱(5%CO2浓度)培养;
3)培养72小时后收集细胞和上清,在37℃水浴和干冰-乙醇浴中反复冻融三次(解冻与冻结每次各2分钟),10000g离心10分钟后上清为AAV粗提液。
AAV滴度测定方法:
使用引物FWD ITR(5'-GGAACCCCTAGTGATGGAGTT)(SEQ ID NO:5),REV ITR(5'-CGGCCTCAGTGAGCGA)(SEQ ID NO:6)特异性检测所有类型AAV载体ITR的序列。
(1)Dnase I消化样品
反应体系成分列在下表中。
取5μL样品,稀释20倍。分相应数目的PCR管,每管18μL消化液,分别加入2μL稀释后样品、和2μL用于制作标准曲线的质粒标准品(含4E+8AAV拷贝(Genome Copies,GC),作为DNase消化对照),相当于稀释10倍,37℃孵育30min。消化后,取5μL样品加入95μL水中,连续稀释2次,共计稀释80000倍,RefAAV(质粒标准品)共计稀释4000倍。
(2)SYBR GreenqPCR
标准品准备:取含2E+8AAV拷贝数/μL的质粒标准品,按8μL+72μL水做6个连续稀释。则第一个梯度浓度为2E+8GC/μL,将其在软件中浓度设置为8E+14GC/mL,以反映样品的稀释梯度。后面系列的梯度依次为8E+13GC/mL,8E+12GC/mL,8E+11GC/mL,8E+10GC/mL,8E+9GC/mL。
反应体系成分列在下表中。
Reagent | Vol.per reaction |
SYBR PCR试剂(2X) | 10μL |
ROX(50X) | 0.4 |
FWD ITR(50μM) | 0.1μL |
REV ITR(50μM) | 0.1μL |
Nuclease-free water | 4.4μL |
样品DNA | 5μL |
按每个样品做3个重复孔计算,配制相应体积的混合物,每孔分18μL,再各自加入2μL样品。
(3)SYBR Green qPCR条件
预变性:95℃ 10min
循环:40cycle:95℃ 15sec;60℃ 1min
全部滴度检测结果转换为与对照载体P5+RC的倍数关系后如图3所示。结果表明,与单独的P5启动子驱动的Rep2Cap2载体“P5+RC”相比,含有DA’或AD’序列的辅助质粒具有增加rAAV病毒产量的作用。
实施例中采用了具体血清型(Rep2Cap2)的辅助质粒,本领域技术人员应理解的是,本发明并不限于该具体血清型,而是可利用目前已知和未来可能继续发现的其他血清型辅助质粒来实施本发明。
本文提及的一些核酸序列如下。
质粒骨架序列(SEQ ID NO:1)
aagccgaattctgcagatatccatcacactggcggccgctcgactagagcggccgccaccgcggtggagctccagcttttgttccctttagtgagggttaattgcgcgcttggcgtaatcatggtcatagctgtttcctgtgtgaaattgttatccgctcacaattccacacaacatacgagccggaagcataaagtgtaaagcctggggtgcctaatgagtgagctaactcacattaattgcgttgcgctcactgcccgctttccagtcgggaaacctgtcgtgccagctgcattaatgaatcggccaacgcgcggggagaggcggtttgcgtattgggcgctcttccgcttcctcgctcactgactcgctgcgctcggtcgttcggctgcggcgagcggtatcagctcactcaaaggcggtaatacggttatccacagaatcaggggataacgcaggaaagaacatgtgagcaaaaggccagcaaaaggccaggaaccgtaaaaaggccgcgttgctggcgtttttccataggctccgcccccctgacgagcatcacaaaaatcgacgctcaagtcagaggtggcgaaacccgacaggactataaagataccaggcgtttccccctggaagctccctcgtgcgctctcctgttccgaccctgccgcttaccggatacctgtccgcctttctcccttcgggaagcgtggcgctttctcatagctcacgctgtaggtatctcagttcggtgtaggtcgttcgctccaagctgggctgtgtgcacgaaccccccgttcagcccgaccgctgcgccttatccggtaactatcgtcttgagtccaacccggtaagacacgacttatcgccactggcagcagccactggtaacaggattagcagagcgaggtatgtaggcggtgctacagagttcttgaagtggtggcctaactacggctacactagaagaacagtatttggtatctgcgctctgctgaagccagttaccttcggaaaaagagttggtagctcttgatccggcaaacaaaccaccgctggtagcggtggtttttttgtttgcaagcagcagattacgcgcagaaaaaaaggatctcaagaagatcctttgatcttttctacggggtctgacgctcagtggaacgaaaactcacgttaagggattttggtcatgagattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgagacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctaaattgtaagcgttaatattttgttaaaattcgcgttaaatttttgttaaatcagctcattttttaaccaataggccgaaatcggcaaaatcccttataaatcaaaagaatagaccgagatagggttgagtgttgttccagtttggaacaagagtccactattaaagaacgtggactccaacgtcaaagggcgaaaaaccgtctatcagggcgatggcccactacgtgaaccatcaccctaatcaagttttttggggtcgaggtgccgtaaagcactaaatcggaaccctaaagggagcccccgatttagagcttgacggggaaagccggcgaacgtggcgagaaaggaagggaagaaagcgaaaggagcgggcgctagggcgctggcaagtgtagcggtcacgctgcgcgtaaccaccacacccgccgcgcttaatgcgccgctacagggcgcgtcccattcgccattcaggctgcgcaactgttgggaagggcgatcggtgcgggcctcttcgctattacgccagctggcgaaagggggatgtgctgcaaggcgattaagttgggtaacgccagggttttcccagtcacgacgttgtaaaacgacggccagtgagcgcgcgtaatacgactcactatagggcgaattgggtaccgggccccccctcgatcgaggtcgacggtatcgggggagct
P5启动子序列(SEQ ID NO:2)
aggtcctgtattagaggtcacgtgagtgttttgcgacattttgcgacaccatgtggtcacgctgggtatttaagcccgagtgagcacgcagggtctccattttgaagcgggaggtttgaacgcgcagccgcc
AD’序列(SEQ ID NO:3)
gcctcagtgagcgagcgagcgcgcagagagggagtggccaactccatcactaggggttcct
DA’序列(SEQ ID NO:4)
aggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggc
参考文献:
1.Xiao X,Li J,Samulski RJ.Production of high-titer recombinant adeno-associated virus vectors in the absence of helper adenovirus.J Virol.1998Mar;72(3):2224-32.doi:10.1128/JVI.72.3.2224-2232.1998.PMID:9499080;PMCID:PMC109519.
2.Flotte TR,Afione SA,Solow R,Drumm ML,Markakis D,Guggino WB,ZeitlinPL,Carter BJ.Expression of the cystic fibrosis transmembrane conductanceregulator from a novel adeno-associated virus promoter.J Biol Chem.1993Feb15;268(5):3781-90.PMID:7679117.
3.Zhang L,Wang DH,Fischer H,Fan PD,Widdicombe JH,Kan YW,DongJY.Efficient expression of CFTR function with adeno-associated virus vectorsthat carry shortened CFTR genes.PNAS.1998August 18;95(17)10158-10163.
SEQUENCE LISTING
<110> 广州派真生物技术有限公司
<120> 制备重组腺相关病毒的辅助质粒及其应用
<130> P10671-I
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 2971
<212> DNA
<213> 人工序列
<220>
<223> 质粒骨架序列
<400> 1
aagccgaatt ctgcagatat ccatcacact ggcggccgct cgactagagc ggccgccacc 60
gcggtggagc tccagctttt gttcccttta gtgagggtta attgcgcgct tggcgtaatc 120
atggtcatag ctgtttcctg tgtgaaattg ttatccgctc acaattccac acaacatacg 180
agccggaagc ataaagtgta aagcctgggg tgcctaatga gtgagctaac tcacattaat 240
tgcgttgcgc tcactgcccg ctttccagtc gggaaacctg tcgtgccagc tgcattaatg 300
aatcggccaa cgcgcgggga gaggcggttt gcgtattggg cgctcttccg cttcctcgct 360
cactgactcg ctgcgctcgg tcgttcggct gcggcgagcg gtatcagctc actcaaaggc 420
ggtaatacgg ttatccacag aatcagggga taacgcagga aagaacatgt gagcaaaagg 480
ccagcaaaag gccaggaacc gtaaaaaggc cgcgttgctg gcgtttttcc ataggctccg 540
cccccctgac gagcatcaca aaaatcgacg ctcaagtcag aggtggcgaa acccgacagg 600
actataaaga taccaggcgt ttccccctgg aagctccctc gtgcgctctc ctgttccgac 660
cctgccgctt accggatacc tgtccgcctt tctcccttcg ggaagcgtgg cgctttctca 720
tagctcacgc tgtaggtatc tcagttcggt gtaggtcgtt cgctccaagc tgggctgtgt 780
gcacgaaccc cccgttcagc ccgaccgctg cgccttatcc ggtaactatc gtcttgagtc 840
caacccggta agacacgact tatcgccact ggcagcagcc actggtaaca ggattagcag 900
agcgaggtat gtaggcggtg ctacagagtt cttgaagtgg tggcctaact acggctacac 960
tagaagaaca gtatttggta tctgcgctct gctgaagcca gttaccttcg gaaaaagagt 1020
tggtagctct tgatccggca aacaaaccac cgctggtagc ggtggttttt ttgtttgcaa 1080
gcagcagatt acgcgcagaa aaaaaggatc tcaagaagat cctttgatct tttctacggg 1140
gtctgacgct cagtggaacg aaaactcacg ttaagggatt ttggtcatga gattatcaaa 1200
aaggatcttc acctagatcc ttttaaatta aaaatgaagt tttaaatcaa tctaaagtat 1260
atatgagtaa acttggtctg acagttacca atgcttaatc agtgaggcac ctatctcagc 1320
gatctgtcta tttcgttcat ccatagttgc ctgactcccc gtcgtgtaga taactacgat 1380
acgggagggc ttaccatctg gccccagtgc tgcaatgata ccgcgagacc cacgctcacc 1440
ggctccagat ttatcagcaa taaaccagcc agccggaagg gccgagcgca gaagtggtcc 1500
tgcaacttta tccgcctcca tccagtctat taattgttgc cgggaagcta gagtaagtag 1560
ttcgccagtt aatagtttgc gcaacgttgt tgccattgct acaggcatcg tggtgtcacg 1620
ctcgtcgttt ggtatggctt cattcagctc cggttcccaa cgatcaaggc gagttacatg 1680
atcccccatg ttgtgcaaaa aagcggttag ctccttcggt cctccgatcg ttgtcagaag 1740
taagttggcc gcagtgttat cactcatggt tatggcagca ctgcataatt ctcttactgt 1800
catgccatcc gtaagatgct tttctgtgac tggtgagtac tcaaccaagt cattctgaga 1860
atagtgtatg cggcgaccga gttgctcttg cccggcgtca atacgggata ataccgcgcc 1920
acatagcaga actttaaaag tgctcatcat tggaaaacgt tcttcggggc gaaaactctc 1980
aaggatctta ccgctgttga gatccagttc gatgtaaccc actcgtgcac ccaactgatc 2040
ttcagcatct tttactttca ccagcgtttc tgggtgagca aaaacaggaa ggcaaaatgc 2100
cgcaaaaaag ggaataaggg cgacacggaa atgttgaata ctcatactct tcctttttca 2160
atattattga agcatttatc agggttattg tctcatgagc ggatacatat ttgaatgtat 2220
ttagaaaaat aaacaaatag gggttccgcg cacatttccc cgaaaagtgc cacctaaatt 2280
gtaagcgtta atattttgtt aaaattcgcg ttaaattttt gttaaatcag ctcatttttt 2340
aaccaatagg ccgaaatcgg caaaatccct tataaatcaa aagaatagac cgagataggg 2400
ttgagtgttg ttccagtttg gaacaagagt ccactattaa agaacgtgga ctccaacgtc 2460
aaagggcgaa aaaccgtcta tcagggcgat ggcccactac gtgaaccatc accctaatca 2520
agttttttgg ggtcgaggtg ccgtaaagca ctaaatcgga accctaaagg gagcccccga 2580
tttagagctt gacggggaaa gccggcgaac gtggcgagaa aggaagggaa gaaagcgaaa 2640
ggagcgggcg ctagggcgct ggcaagtgta gcggtcacgc tgcgcgtaac caccacaccc 2700
gccgcgctta atgcgccgct acagggcgcg tcccattcgc cattcaggct gcgcaactgt 2760
tgggaagggc gatcggtgcg ggcctcttcg ctattacgcc agctggcgaa agggggatgt 2820
gctgcaaggc gattaagttg ggtaacgcca gggttttccc agtcacgacg ttgtaaaacg 2880
acggccagtg agcgcgcgta atacgactca ctatagggcg aattgggtac cgggcccccc 2940
ctcgatcgag gtcgacggta tcgggggagc t 2971
<210> 2
<211> 132
<212> DNA
<213> 人工序列
<220>
<223> 启动子序列
<400> 2
aggtcctgta ttagaggtca cgtgagtgtt ttgcgacatt ttgcgacacc atgtggtcac 60
gctgggtatt taagcccgag tgagcacgca gggtctccat tttgaagcgg gaggtttgaa 120
cgcgcagccg cc 132
<210> 3
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> AD'序列
<400> 3
gcctcagtga gcgagcgagc gcgcagagag ggagtggcca actccatcac taggggttcc 60
t 61
<210> 4
<211> 61
<212> DNA
<213> 人工序列
<220>
<223> DA'序列
<400> 4
aggaacccct agtgatggag ttggccactc cctctctgcg cgctcgctcg ctcactgagg 60
c 61
<210> 5
<211> 21
<212> DNA
<213> 人工序列
<220>
<223> 引物序列
<400> 5
ggaaccccta gtgatggagt t 21
<210> 6
<211> 16
<212> DNA
<213> 人工序列
<220>
<223> 引物序列
<400> 6
cggcctcagt gagcga 16
Claims (10)
1.用于制备重组腺相关病毒的辅助质粒,包括:
1)AAV的Rep和Cap蛋白编码序列;
2)至少一个启动子序列;以及
3)至少一个DA’序列或DA’序列的反向互补序列AD’序列。
2.如权利要求1所述的辅助质粒,其中至少一个DA’或AD’序列位于Rep和Cap蛋白编码序列的上游。
3.如权利要求1或2所述的辅助质粒,其中至少一个DA’或AD’序列位于所述Rep和Cap蛋白编码序列的下游。
4.如权利要求1-3任一项所述的辅助质粒,其中,所述辅助质粒从5’至3’方向依次包括:
i)DA’或AD’序列、启动子序列以及Rep和Cap蛋白编码序列;
ii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列以及DA’或AD’序列;
iii)DA’或AD’序列、启动子序列、Rep和Cap蛋白编码序列、DA’或AD’序列以及启动子序列;或
iv)Rep和Cap蛋白编码序列、DA’或AD’序列以及启动子序列。
5.如权利要求1-4任一项所述的辅助质粒,其中所述启动子为P5启动子。
6.如权利要求1-5任一项所述的辅助质粒,其中所述P5启动子序列包括SEQ ID NO:2所示序列或与SEQ ID NO:2所示序列具有至少90%序列一致性的功能性变体。
7.如权利要求1-6任一项所述的辅助质粒,其中所述DA’序列包括SEQ ID NO:4所示序列或为与SEQ ID NO:4所示序列具有至少90%序列一致性的功能性变体。
8.如权利要求1-7任一项所述的辅助质粒,其中所述AD’序列包括SEQ ID NO:3所示序列或为与SEQ ID NO:3所示序列具有至少90%序列一致性的功能性变体。
9.如权利要求1-8任一项所述的辅助质粒,其中所述Rep和Cap蛋白来自2型腺相关病毒。
10.提高重组腺相关病毒生产能力的方法,包括使用权利要求1-9任一项所述的辅助质粒转化宿主细胞。
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WO2023143063A1 (zh) * | 2022-01-25 | 2023-08-03 | 广州派真生物技术有限公司 | 一种提高杆状病毒系统生产腺相关病毒的方法及应用 |
CN117660532A (zh) * | 2023-12-13 | 2024-03-08 | 广州派真生物技术有限公司 | 一种降低重组腺相关病毒中rcAAV残留的辅助质粒及应用 |
CN117683797A (zh) * | 2023-12-04 | 2024-03-12 | 广州派真生物技术有限公司 | 一种用于重组腺相关病毒包装的质粒系统及其应用 |
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US20240076319A1 (en) | 2024-03-07 |
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