Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10
Prolongation of sheep corneal allograft survival by ex vivo transfer of the gene encoding interleukin-10
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DOI：
10.1097/00007890-200105150-00006
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发表时间：
2001-05-15
期刊：
影响因子：
6.2
通讯作者：
Williams, KA
中科院分区：
文献类型：
--
作者：
Klebe, S;Sykes, PJ;Williams, KA
背景 通过离体基因治疗对供体角膜进行修饰有可能调节不可逆排斥反应，这是角膜移植失败的主要原因。我们的目的是将编码哺乳动物 IL-10 的基因转移到绵羊供体角膜，并确定随后的原位角膜同种异体移植物在远交绵羊模型中的存活率。方法。故意损伤后通过放射自显影测定绵羊角膜内皮的复制能力。使用复制缺陷型腺病毒将lacZ报告基因递送至绵羊角膜，并在体外对转染的角膜进行器官培养，以确定转染效率、报告基因表达的持续时间以及归因于载体的毒性。将编码全长绵羊IL-10的cDNA克隆到腺病毒载体中，该载体用于在移植前离体转染供体角膜，在远交羊中进行原位穿透性角膜移植。结果。发现绵羊角膜内皮基本上是无丝分裂的，用病毒载体实现了> 70%的角膜内皮细胞的转染，并且表达在体外维持28天。在体外器官培养的转染角膜中可检测到 IL-10 mRNA 21 天。与对照角膜（中位20.5天，范围18-32天，P=0.011）相比，用编码IL-10的cDNA转染的供体角膜在穿透性角膜移植术后显示出显着延长的存活期（中位55天，范围19大于或等于300天）。结论。局部基因治疗介导免疫调节细胞因子 IL-10 的表达有可能降低角膜移植排斥的发生率并延长同种异体角膜移植物的存活率。
Background Modification of a donor cornea by gene therapy ex vivo has potential to modulate irreversible rejection, the major cause of corneal graft failure. Our aim was to transfer the gene encoding mammalian IL-10 to ovine donor corneas and to determine subsequent, orthotopic corneal allograft survival in an outbred sheep model.Methods. The replicative capacity of ovine corneal endothelium was determined by autoradiography after deliberate injury. A replication defective adenovirus was used to deliver the lacZ reporter gene to ovine corneas and transfected corneas were organ-cultured in vitro to allow transfection efficiency, duration of reporter gene expression, and toxicity attributable to the vector to be determined. A cDNA encoding full-length ovine IL-10 was cloned into an adenoviral vector that was used to transfect donor corneas ex vivo before transplantation, Orthotopic penetrating corneal transplantation was performed in outbred sheep.Results. Sheep corneal endothelium was found to be essentially amitotic, Transfection of > 70% corneal endothelial cells was achieved with the viral vector and expression was maintained for 28 days in vitro. IL-10 mRNA was detectable in transfected, organ-cultured corneas for 21 days in vitro. Donor corneas transfected with cDNA encoding IL-10 showed significantly prolonged survival after penetrating keratoplasty (median 55 days, range 19 greater than or equal to 300 days) compared with control corneas (median 20.5 days, range 18-32 days, P=0.011).Conclusion. Local gene therapy mediated expression of the immunomodulatory cytokine IL-10 has the potential to reduce the incidence of corneal graft rejection and to prolong corneal allograft survival.