Nakai H, Yant S R, Storm T A, Fuess S, Meuse L, Kay M A
Department of Pediatrics, Stanford University School of Medicine, Stanford, California 94305, USA.
J Virol. 2001 Aug;75(15):6969-76. doi: 10.1128/JVI.75.15.6969-6976.2001.
Recombinant adeno-associated virus (rAAV) vectors stably transduce hepatocytes in experimental animals. Although the vector genomes are found both as extrachromosomes and as chromosomally integrated forms in hepatocytes, the relative proportion of each has not yet been clearly established. Using an in vivo assay based on the induction of hepatocellular regeneration via a surgical two-thirds partial hepatectomy, we have determined the proportion of integrated and extrachromosomal rAAV genomes in mouse livers and their relative contribution to stable gene expression in vivo. Plasma human coagulation factor IX (hF.IX) levels in mice originating from a chromosomally integrated hF.IX-expressing transposon vector remained unchanged with hepatectomy. This was in sharp contrast to what was observed when a surgical partial hepatectomy was performed in mice 6 weeks to 12 months after portal vein injection of a series of hF.IX-expressing rAAV vectors. At doses of 2.4 x 10(11) to 3.0 x 10(11) vector genomes per mouse (n = 12), hF.IX levels and the average number of stably transduced vector genomes per cell decreased by 92 and 86%, respectively, after hepatectomy. In a separate study, one of three mice injected with a higher dose of rAAV had a higher proportion (67%) of integrated genomes, the significance of which is not known. Nevertheless, in general, these results indicate that, in most cases, no more than approximately 10% of stably transduced genomes integrated into host chromosomes in vivo. Additionally, the results demonstrate that extrachromosomal, not integrated, genomes are the major form of rAAV in the liver and are the primary source of rAAV-mediated gene expression. This small fraction of integrated genomes greatly decreases the potential risk of vector-related insertional mutagenesis associated with all integrating vectors but also raises uncertainties as to whether rAAV-mediated hepatic gene expression can persist lifelong after a single vector administration.
重组腺相关病毒（rAAV）载体可在实验动物中稳定转导肝细胞。虽然在肝细胞中发现载体基因组既以染色体外形式存在，也以染色体整合形式存在，但每种形式的相对比例尚未明确确定。通过基于手术三分之二部分肝切除术诱导肝细胞再生的体内试验，我们确定了小鼠肝脏中整合型和染色体外rAAV基因组的比例及其对体内稳定基因表达的相对贡献。源自染色体整合的表达人凝血因子IX（hF.IX）的转座子载体的小鼠血浆中hF.IX水平在肝切除术后保持不变。这与在门静脉注射一系列表达hF.IX的rAAV载体6周至12个月后对小鼠进行手术部分肝切除术时观察到的情况形成鲜明对比。在每只小鼠注射2.4×10¹¹至3.0×x10¹¹载体基因组的剂量下（n = 12），肝切除术后hF.IX水平和每个细胞中稳定转导的载体基因组平均数量分别下降了92%和86%。在另一项研究中，三只注射更高剂量rAAV的小鼠中有一只整合基因组的比例更高（67%），其意义尚不清楚。然而，总体而言，这些结果表明，在大多数情况下，体内稳定转导的基因组中不超过约10%整合到宿主染色体中。此外，结果表明，染色体外而非整合的基因组是肝脏中rAAV的主要形式，并且是rAAV介导的基因表达的主要来源。这一小部分整合基因组大大降低了与所有整合载体相关的载体相关插入诱变的潜在风险，但也引发了关于rAAV介导的肝脏基因表达在单次载体给药后是否能终身持续的不确定性。
