Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males
Lack of germline transmission of vector sequences following systemic administration of recombinant AAV-2 vector in males
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DOI：
10.1006/mthe.2001.0491
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发表时间：
2001-12-01
影响因子：
12.4
通讯作者：
High, KA
中科院分区：
文献类型：
--
作者：
Arruda, VR;Fields, PA;High, KA
病毒载体全身给药的一个潜在后果是外源DNA意外引入受体生殖细胞。为了评估体内重组腺相关病毒（rAAV）介导的血友病B基因转移方法的安全性，我们探究了在四种雄性动物（小鼠、大鼠、兔和狗）肌肉内（IM）注射rAAV后载体序列生殖系传递的风险。在小鼠和大鼠的载体生物分布研究中，使用灵敏的PCR技术在性腺DNA中检测到载体序列的可能性呈剂量依赖性增加。然而，从狗的精液中提取的DNA对载体序列呈阴性。为了解决这一差异，在兔中进行了研究，并对精液和睾丸DNA进行了载体序列存在情况的分析。这些研究表明，在每千克肌肉内注射1×10¹³载体基因组rAAV（vg）后7至90天内收集的兔精液样本中提取的DNA未检测到AAV载体序列。相反，从性腺组织中提取的DNA对载体序列呈阳性，但阳性信号的数量和强度随时间减弱。通过荧光原位杂交（FISH）分析，AAV信号定位于睾丸基底膜和间质空间；未观察到细胞内信号。我们在大鼠和狗的肝动脉注射rAAV后也观察到了类似的结果，这表明我们的发现与载体的给药途径无关。试图用AAV - lacZ直接转导分离的小鼠精原细胞未成功。在临床研究中，肌肉内注射剂量高达2×10¹² vg/kg的AAV载体的人类受试者的精液中未显示出载体序列的证据。总之，这些研究表明，引入骨骼肌或肝动脉的rAAV不能有效地转导雄性生殖细胞。我们得出结论，在肌肉内或肝动脉注射AAV - 2载体后载体序列意外生殖系传递的风险极低。
A potential consequence of systemic administration of viral vectors is the inadvertent introduction of foreign DNA into recipient germ cells. To evaluate the safety of in vivo recombinant adeno-associated virus (rAAV) mediated gene transfer approaches for hemophilia B, we explored the risk of germline transmission of vector sequences following intramuscular (IM) injection of rAAV in four species of male animals (mouse, rat, rabbit and dog). In vector biodistribution studies in mice and rats, there is a dose-dependent increase in the likelihood that vector sequences can be detected in gonadal DNA using a sensitive PCR technique. However, in dogs DNA extracted from semen is negative for vector sequences. To address this discrepancy, studies were done in rabbits, and both semen and testicular DNAs were analyzed for the presence of vector sequences. These studies showed that no AAV vector sequences were detected in DNA extracted from rabbit semen samples collected at time points ranging from 7 to 90 days following IM injection of 1 x 10(13) vector genomes rAAV (vg) per kg. In contrast, DNA extracted from gonadal tissue was positive for vector sequences, but the positive signals diminished in number and strength with time. By FISH analysis, AAV signals were localized to the testis basement membrane and the interstitial space; no intracellular signal was observed. We observed similar findings following hepatic artery administration of rAAV in rats and dogs, suggesting that our findings are independent of the route of administration of vector. Attempts to transduce isolated murine spermatogonia directly with AAV-lacZ were unsuccessful. In clinical studies human subjects injected IM with an AAV vector at doses up to 2 x 10(12) vg/kg have shown no evidence of vector sequences in semen. Together, these studies suggest that rAAV introduced into skeletal muscle or the hepatic artery does not transduce male germ cells efficiently. We conclude that the risk of inadvertent germline transmission of vector sequences following IM or hepatic artery injection of AAV-2 vectors is extremely low.