Inagaki Katsuya, Piao Chuncheng, Kotchey Nicole M, Wu Xiaolin, Nakai Hiroyuki
Department of Microbiology and Molecular Genetics, University of Pittsburgh School of Medicine, W1244 BSTWR, 200 Lothrop Street, Pittsburgh, PA 15261, USA.
J Virol. 2008 Oct;82(19):9513-24. doi: 10.1128/JVI.01001-08. Epub 2008 Jul 9.
Neonatal injection of recombinant adeno-associated virus serotype 8 (rAAV8) vectors results in widespread transduction in multiple organs and therefore holds promise in neonatal gene therapy. On the other hand, insertional mutagenesis causing liver cancer has been implicated in rAAV-mediated neonatal gene transfer. Here, to better understand rAAV integration in neonatal livers, we investigated the frequency and spectrum of genomic integration of rAAV8 vectors in the liver following intraperitoneal injection of 2.0 x 10(11) vector genomes at birth. This dose was sufficient to transduce a majority of hepatocytes in the neonatal period. In the first approach, we injected mice with a beta-galactosidase-expressing vector at birth and quantified rAAV integration events by taking advantage of liver regeneration in a chronic hepatitis animal model and following partial hepatectomy. In the second approach, we performed a new, quantitative rAAV vector genome rescue assay by which we identified rAAV integration sites and quantified integrations. As a result, we find that at least approximately 0.05% of hepatocytes contained rAAV integration, while the average copy number of integrated double-stranded vector genome per cell in the liver was approximately 0.2, suggesting concatemer integration. Twenty-three of 34 integrations (68%) occurred in genes, but none of them were near the mir-341 locus, the common rAAV integration site found in mouse hepatocellular carcinoma. Thus, rAAV8 vector integration occurs preferentially in genes at a frequency of 1 in approximately 10(3) hepatocytes when a majority of hepatocytes are once transduced in the neonatal period. Further studies are warranted to elucidate the relationship between vector dose and integration frequency or spectrum.
新生期注射重组8型腺相关病毒（rAAV8）载体可导致多个器官广泛转导，因此在新生期基因治疗中具有前景。另一方面，rAAV介导的新生期基因转移与导致肝癌的插入诱变有关。在此，为了更好地了解rAAV在新生期肝脏中的整合情况，我们研究了出生时腹腔注射2.0×10¹¹载体基因组后rAAV8载体在肝脏中的基因组整合频率和谱。该剂量足以在新生期转导大多数肝细胞。在第一种方法中，我们在出生时给小鼠注射表达β-半乳糖苷酶的载体，并利用慢性肝炎动物模型中的肝脏再生以及部分肝切除术后的情况来量化rAAV整合事件。在第二种方法中，我们进行了一种新的定量rAAV载体基因组拯救分析，通过该分析我们鉴定了rAAV整合位点并对整合进行了量化。结果，我们发现至少约0.05%的肝细胞含有rAAV整合，而肝脏中每个细胞整合的双链载体基因组的平均拷贝数约为0.2，提示串联整合。34个整合中有23个（68%）发生在基因中，但它们均不在mir-341位点附近，mir-341位点是在小鼠肝细胞癌中发现的常见rAAV整合位点。因此，当在新生期大多数肝细胞被转导一次时，rAAV8载体整合优先以大约每10³个肝细胞中有1个的频率发生在基因中。有必要进行进一步研究以阐明载体剂量与整合频率或谱之间的关系。
