Spliceosome-mediated RNA trans-splicing enables correction or labeling of pre-mRNA, but therapeutic applications are hampered by issues related to the activity and target specificity of trans-splicing RNA (tsRNA). We employed computational RNA structure design to improve both on-target activity and specificity of tsRNA in a herpes simplex virus thymidine kinase/ganciclovir suicide gene therapy approach targeting alpha fetoprotein (AFP), a marker of hepatocellular carcinoma (HCC) or human papillomavirus type 16 (HPV-16) pre-mRNA. While unstructured, mismatched target binding domains significantly improved 3′ exon replacement (3’ER), 5′ exon replacement (5’ER) correlated with the thermodynamic stability of the tsRNA 3′ end. Alternative on-target trans-splicing was found to be a prevalent event. The specificity of trans-splicing with the intended target splice site was improved 10-fold by designing tsRNA that harbors secondary target binding domains shielding alternative on-target and blinding off-target splicing events. Such rationally designed suicide RNAs efficiently triggered death of HPV-16-transduced or hepatoblastoma-derived human tissue culture cells without evidence for off-target cell killing. Highest cell death activities were observed with novel dual-targeting tsRNAs programmed for trans-splicing toward AFP and a second HCC pre-mRNA biomarker. Our observations suggest trans-splicing represents a promising approach to suicide gene therapy.

剪接体介导的RNA反式剪接能够校正或标记前mRNA，但与反式剪接RNA（tsRNA）的活性和靶标特异性相关的问题阻碍了治疗应用。我们采用计算性RNA结构设计来改善针对单纯性疱疹病毒胸苷激酶/更昔洛韦自杀基因治疗方法中靶向甲胎蛋白（AFP），肝细胞癌（HCC）或人乳头瘤病毒16型标记物的tsRNA的靶向活性和特异性（HPV-16）pre-mRNA。虽然是非结构化的，但错配的靶结合域显着改善了3'外显子置换（3'ER），5'外显子置换（5'ER）与tsRNA 3'末端的热力学稳定性有关。替代性目标转移-拼接被发现是一个普遍的事件。通过设计tsRNA，将带有预期靶标剪接位点的反式剪接的特异性提高了10倍，该tsRNA带有二级靶标结合结构域，可屏蔽替代的靶上靶和盲靶脱靶剪接事件。这种合理设计的自杀性RNA有效触发了HPV-16转导的或肝母细胞瘤衍生的人类组织培养细胞的死亡，而没有证据表明脱靶细胞会被杀死。用新颖的双重靶向tsRNAs观察到最高的细胞死亡活性，所述tsRNAs被编程用于向AFP反式剪接和第二种HCC pre-mRNA生物标记。我们的观察结果表明，转拼是自杀基因治疗的一种有前途的方法。