Murakami Pete, Pungor Erno, Files Jim, Do Linh, van Rijnsoever Richard, Vogels Ronald, Bout Abraham, McCaman Michael
Berlex Biosciences, Process Development Department, Richmond, CA 94804, USA.
Hum Gene Ther. 2002 May 20;13(8):909-20. doi: 10.1089/10430340252939023.
An undesirable byproduct from recombinant adenoviral vectors is the emergence of replication competent adenovirus (RCA) that result from rare homologous recombination events between the viral E1-containing (permissive) mammalian host cell genome and the virus itself, restoring the E1 gene to the viral genome. To reduce or eliminate the problem of RCA, we evaluated production of a first generation Ad5 vector (Ad5FGF4) in the cell line PER.C6. This E1-transformed human cell line contains only Ad5 nucleotides 459-3510, which precludes double crossover-type homologous recombination because the Ad5FGF-4 only contains 5' Ad5 sequence up to nucleotide 453. The Ad5FGF4 vector does, however, retain 177 nucleotides of the 3' end of the E1B-55K gene that are also present in PER.C6. With only this single region of homology between vector and cell line, we were surprised to detect virus-specific cytopathic effects (CPE) in our cell-based assay for RCA. This CPE-inducing agent was amplified in nonpermissive A549 cells but also supported amplification of the parental Ad5FGF-4. Because we were unable to isolate the CPE-inducing agent in pure form we first identified it as atypical RCA. Polymerase chain reaction (PCR) and Southern blot experiments identified viral DNA segments in which recombination had occurred between the 177 nucleotides of E1B present in both Ad5FGF-4 and PER.C6. The atypical RCA genomes contain a copy of the original (PGK promoter-E1 gene carrying) plasmid used in the construction of the PER.C6 cell line and they retained the parental FGF-4 transgene. However, significant deletions occurred within the recombined genomes in compensation for the large insertion from PER.C6 sequences and resulted in the loss of essential viral genes. This deletion renders these recombinant viruses replication defective, requiring helper functions from remaining parental Ad5FGF-4 for amplification. These atypical RCA entities may be more properly designated as helper-dependent E1-positive particles (HDEPs). This finding shows the importance of avoiding the use of "nonmatched" vectors where any overlap exists between the recombinant vector and E1 sequences in the packaging cell line. The cloning of the FGF-4 transgene into an adenoviral vector specifically "matched" for PER.C6 (lacking the 177 nucleotide region of homology) has allowed extensive virus propagation (Ad5.1FGF-4) with no CPE- or HDEP-like events yet detected.
重组腺病毒载体产生的一种不良副产物是复制型腺病毒（RCA）的出现，它源于含病毒E1区的（允许性）哺乳动物宿主细胞基因组与病毒自身之间罕见的同源重组事件，使E1基因恢复到病毒基因组中。为了减少或消除RCA问题，我们评估了在PER.C6细胞系中生产第一代Ad5载体（Ad5FGF4）的情况。这个E1转化的人类细胞系仅包含Ad5核苷酸459 - 3510，这排除了双交换型同源重组，因为Ad5FGF - 4仅包含直至核苷酸453的5'端Ad5序列。然而，Ad5FGF4载体确实保留了E1B - 55K基因3'端的177个核苷酸，PER.C6细胞系中也存在这些核苷酸。由于载体与细胞系之间仅存在这一个同源区域，我们很惊讶地在基于细胞的RCA检测中检测到病毒特异性细胞病变效应（CPE）。这种诱导CPE的因子在非允许性A549细胞中得以扩增，但也支持亲本Ad5FGF - 4的扩增。由于我们无法以纯形式分离出诱导CPE的因子，我们最初将其鉴定为非典型RCA。聚合酶链反应（PCR）和Southern印迹实验鉴定出了病毒DNA片段，其中Ad5FGF - 4和PER.C6中都存在的E1B的177个核苷酸之间发生了重组。非典型RCA基因组包含构建PER.C6细胞系时使用的原始（携带PGK启动子 - E1基因的）质粒的一个拷贝，并且它们保留了亲本FGF - 4转基因。然而，在重组基因组中发生了显著缺失，以补偿来自PER.C6序列的大插入，导致必需病毒基因的丢失。这种缺失使这些重组病毒复制缺陷，需要剩余亲本Ad5FGF - 4的辅助功能才能扩增。这些非典型RCA实体可能更恰当地被指定为辅助依赖型E1阳性颗粒（HDEP）。这一发现表明，当重组载体与包装细胞系中的E1序列存在任何重叠时，避免使用“不匹配”载体的重要性。将FGF - 4转基因克隆到与PER.C6特异性“匹配”（缺乏177个核苷酸同源区域）的腺病毒载体中，使得病毒能够大量繁殖（Ad5.1FGF - 4），且尚未检测到类似CPE或HDEP的事件。
