Garrett Deiadra J, Larson Janet E, Dunn Daisy, Marrero Luis, Cohen J Craig
Ochsner Children's Research Institute, Ochsner Clinic Foundation, New Orleans, LA 70121, USA.
BMC Biotechnol. 2003 Sep 30;3:16. doi: 10.1186/1472-6750-3-16.
Gene transfer into the amniotic fluid using recombinant adenovirus vectors was shown previously to result in high efficiency transfer of transgenes into the lungs and intestines. Adenovirus mediated in utero gene therapy, however, resulted in expression of the transgene for less than 30 days. Recombinant adenovirus associated viruses (rAAV) have the advantage of maintaining the viral genome in daughter cells thus providing for long-term expression of transgenes.
Recombinant AAV2 carrying green fluorescent protein (GFP) was introduced into the amniotic sac of fetal rodents and nonhuman primates. Transgene maintenance and expression was monitor.
Gene transfer resulted in rapid uptake and long-term gene expression in mice, rats, and non-human primates. Expression and secretion of the reporter gene, GFP, was readily demonstrated within 72 hours post-therapy. In long-term studies in rats and nonhuman primates, maintenance of GFP DNA, protein expression, and reporter gene secretion was documented for over one year.
Because only multipotential stem cells are present at the time of therapy, these data demonstrated that in utero gene transfer with AAV2 into stem cells resulted in long-term systemic expression of active transgene roducts. Thus, in utero gene transfer via the amniotic fluid may be useful in treatment of gene disorders.
先前研究表明，使用重组腺病毒载体将基因导入羊水可实现转基因高效导入肺和肠道。然而，腺病毒介导的子宫内基因治疗导致转基因表达不足30天。重组腺相关病毒（rAAV）具有在子代细胞中维持病毒基因组从而实现转基因长期表达的优势。
将携带绿色荧光蛋白（GFP）的重组AAV2导入胎鼠和非人灵长类动物的羊膜囊。监测转基因的维持和表达情况。
基因转移在小鼠、大鼠和非人灵长类动物中导致快速摄取和长期基因表达。治疗后72小时内即可轻松证明报告基因GFP的表达和分泌。在对大鼠和非人灵长类动物的长期研究中，记录到GFP DNA、蛋白质表达和报告基因分泌持续维持了一年多。
由于治疗时仅存在多能干细胞，这些数据表明，用AAV2进行子宫内基因转移至干细胞可导致活性转基因产物的长期全身表达。因此，通过羊水进行子宫内基因转移可能对治疗基因疾病有用。
