Stanford University, Departments of Pediatrics and Genetics, Stanford, California 94305-5164, USA.
Mol Ther. 2012 Oct;20(10):1912-23. doi: 10.1038/mt.2012.164. Epub 2012 Sep 18.
Although recombinant adeno-associated virus (rAAV) vectors are proving to be efficacious in clinical trials, the episomal character of the delivered transgene restricts their effectiveness to use in quiescent tissues, and may not provide lifelong expression. In contrast, integrating vectors enhance the risk of insertional mutagenesis. In an attempt to overcome both of these limitations, we created new rAAV-rDNA vectors, with an expression cassette flanked by ribosomal DNA (rDNA) sequences capable of homologous recombination into genomic rDNA. We show that after in vivo delivery the rAAV-rDNA vectors integrated into the genomic rDNA locus 8-13 times more frequently than control vectors, providing an estimate that 23-39% of the integrations were specific to the rDNA locus. Moreover, a rAAV-rDNA vector containing a human factor IX (hFIX) expression cassette resulted in sustained therapeutic levels of serum hFIX even after repeated manipulations to induce liver regeneration. Because of the relative safety of integration in the rDNA locus, these vectors expand the usage of rAAV for therapeutics requiring long-term gene transfer into dividing cells.
尽管重组腺相关病毒 (rAAV) 载体在临床试验中被证明是有效的，但所递送的转基因的附加体性质将其有效性限制在静止组织中的使用，并且可能无法提供终身表达。相比之下，整合载体会增加插入突变的风险。为了克服这两个限制，我们创建了新的 rAAV-rDNA 载体，其表达盒两侧为核糖体 DNA(rDNA) 序列，能够通过同源重组整合到基因组 rDNA 中。我们表明，在体内递送后，rAAV-rDNA 载体整合到基因组 rDNA 位点的频率比对照载体高 8-13 倍，估计有 23-39%的整合是针对 rDNA 位点的特异性的。此外，含有人凝血因子 IX (hFIX) 表达盒的 rAAV-rDNA 载体在多次诱导肝再生以进行肝脏再生的操作后，仍能持续保持血清 hFIX 的治疗水平。由于整合到 rDNA 位点的相对安全性，这些载体扩展了 rAAV 在需要长期基因转移到分裂细胞中的治疗中的使用。
