Optimized riboswitch-regulated AAV vector for VEGF-B gene therapy

Frontiers in Medicine

Medicine

Seppo Ylä-Herttuala¹  Sanna K. Peltola²  Igor Oruetxebarria²  Saana Lepola²  Hanna M. Leinonen²  Tiina Nieminen²  Kari J. Airenne²  Eevi M. Lipponen²  Lionel Galibert²  Petra Tikkanen²  Hanna P. Lesch²  Anniina J. Valkama²  Reetta A. E. Eriksson³  Piia Käyhty³ 

[1] A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland;Gene Therapy Unit and Research Center, Kuopio University Hospital, Kuopio, Finland;Kuopio Center for Gene and Cell Therapy, Kuopio, Finland;Kuopio Center for Gene and Cell Therapy, Kuopio, Finland;A.I. Virtanen Institute for Molecular Sciences, University of Eastern Finland, Kuopio, Finland;

关键词: riboswitch;    ON-switch;    gene therapy;    AAV (adeno-associated virus);    VEGF-B;    tetracycline;    transgene expression regulation;   

DOI  :  10.3389/fmed.2022.1052318

 received in 2022-09-23, accepted in 2022-11-24,  发布年份 2022

来源: Frontiers

【 摘 要 】

Gene therapy would greatly benefit from a method to regulate therapeutic gene expression temporally. Riboswitches are small RNA elements that have been studied for their potential use in turning transgene expression on or off by ligand binding. We compared several tetracycline and toyocamycin-inducible ON-riboswitches for a drug responsive transgene expression. The tetracycline-dependent K19 riboswitch showed the best control and we successfully applied it to different transgenes. The induction of gene expression was 6- to 10-fold, dose-dependent, reversible, and occurred within hours after the addition of a clinically relevant tetracycline dose, using either plasmid or adeno-associated virus (AAV) vectors. To enhance the switching capacity, we further optimized the gene cassette to control the expression of a potential therapeutic gene for cardiovascular diseases, VEGF-B. Using two or three riboswitches simultaneously reduced leakiness and improved the dynamic range, and a linker sequence between the riboswitches improved their functionality. The riboswitch function was promoter-independent, but a post-transcriptional WPRE element in the expression cassette reduced its functionality. The optimized construct was a dual riboswitch at the 3′ end of the transgene with a 100 bp linker sequence. Our study reveals significant differences in the function of riboswitches and provides important aspects on optimizing expression cassette designs. The findings will benefit further research and development of riboswitches.

【 授权许可】

Unknown   
Copyright © 2022 Eriksson, Nieminen, Galibert, Peltola, Tikkanen, Käyhty, Leinonen, Oruetxebarria, Lepola, Valkama, Lipponen, Lesch, Ylä-Herttuala and Airenne.