Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.
Querying Recombination Junctions of Replication-Competent Adeno-Associated Viruses in Gene Therapy Vector Preparations with Single Molecule, Real-Time Sequencing.
复制标题
DOI：
10.3390/v15061228
复制
发表时间：
2023-05-24
期刊：
影响因子：
--
通讯作者：
Tai PWL
中科院分区：
文献类型：
--
作者：
Yip M;Chen J;Zhi Y;Tran NT;Namkung S;Pastor E;Gao G;Tai PWL
用于基因治疗的腺相关病毒（AAV）载体的临床级制剂通常经历一系列诊断以确定滴度、纯度、均一性和DNA污染物的存在。一种类型的污染物，仍然研究不足的是复制能力（rc）的AAV。rcAAV通过源自生产材料的DNA重组形成，产生完整的、可复制的和潜在感染性的病毒样病毒体。它们可以通过在野生型腺病毒存在下将来自用AAV载体转导的细胞的裂解物连续传代来检测。对最后一次传代的细胞裂解物进行qPCR，以检测rep基因的存在。不幸的是，该方法不能用于查询重组事件的多样性，qPCR也不能提供rcAAV如何产生的见解。因此，通过ITR侧翼的目的基因（GOI）构建体和携带rep-cap基因的表达构建体之间的错误重组事件形成rcAAV的描述很少。我们已经使用单分子实时测序（SMRT）来分析从rcAAV阳性载体制备物扩增的病毒样基因组。我们目前的证据表明，序列独立和非同源重组ITR轴承转基因和rep/cap质粒之间发生在几个事件和rcAAV产卵从不同的克隆。
Clinical-grade preparations of adeno-associated virus (AAV) vectors used for gene therapy typically undergo a series of diagnostics to determine titer, purity, homogeneity, and the presence of DNA contaminants. One type of contaminant that remains poorly investigated is replication-competent (rc)AAVs. rcAAVs form through recombination of DNA originating from production materials, yielding intact, replicative, and potentially infectious virus-like virions. They can be detected through the serial passaging of lysates from cells transduced by AAV vectors in the presence of wildtype adenovirus. Cellular lysates from the last passage are subjected to qPCR to detect the presence of the rep gene. Unfortunately, the method cannot be used to query the diversity of recombination events, nor can qPCR provide insights into how rcAAVs arise. Thus, the formation of rcAAVs through errant recombination events between ITR-flanked gene of interest (GOI) constructs and expression constructs carrying the rep-cap genes is poorly described. We have used single molecule, real-time sequencing (SMRT) to analyze virus-like genomes expanded from rcAAV-positive vector preparations. We present evidence that sequence-independent and non-homologous recombination between the ITR-bearing transgene and the rep/cap plasmid occurs under several events and rcAAVs spawn from diverse clones.
登录
查看更多内容
RAAV矢量中的亚基因组颗粒是由DNA病变/断裂和载体基因组的非同源末端连接而产生的。
DOI：
10.1016/j.omtn.2022.08.027
发表时间：
2022-09-13
期刊：
MOLECULAR THERAPY NUCLEIC ACIDS
影响因子：
--
作者：
Zhang, Junping;Guo, Ping;Yu, Xiangping;Frabutt, Dylan A.;Lam, Anh K.;Mulcrone, Patrick L.;Chrzanowski, Matthew;Firrman, Jenni;Pouchnik, Derek;Sang, Nianli;Diao, Yong;Herzog, Roland W.;Xiao, Weidong
通讯作者：
Xiao, Weidong
一项对患有 A 型血友病的狗进行 AAV 基因治疗的长期研究确定了转导肝细胞的克隆扩增。
影响因子：
46.9
作者：
Nguyen GN;Everett JK;Kafle S;Roche AM;Raymond HE;Leiby J;Wood C;Assenmacher CA;Merricks EP;Long CT;Kazazian HH;Nichols TC;Bushman FD;Sabatino DE
通讯作者：
Sabatino DE
影响因子：
4.2
作者：
Yan, Ziying;Sun, Xingshen;Engelhardt, John F.
通讯作者：
Engelhardt, John F.
影响因子：
12.4
作者：
Sabatino, Denise E.;Bushman, Frederic D.;Chandler, Randy J.;Crystal, Ronald G.;Davidson, Beverly L.;Dolmetsch, Ricardo;Eggan, Kevin C.;Gao, Guangping;Gil-Farina, Irene;Kay, Mark A.;McCarty, Douglas M.;Montini, Eugenio;Ndu, Adora;Yuan, Jing
通讯作者：
Yuan, Jing
影响因子：
5.4
作者：
Allen, JM;Debelak, DJ;Miller, AD
通讯作者：
Miller, AD