Song Yuhu, Lou Howard H, Boyer Julie L, Limberis Maria P, Vandenberghe Luk H, Hackett Neil R, Leopold Philip L, Wilson James M, Crystal Ronald G
Department of Genetic Medicine, Weill Cornell Medical College, New York, NY 10065, USA.
Hum Gene Ther. 2009 Mar;20(3):267-81. doi: 10.1089/hum.2008.173.
Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.
囊性纤维化的特征是囊性纤维化跨膜传导调节因子（CFTR）缺乏，CFTR是一种氯离子转运蛋白。腺相关病毒（AAV）载体的包装限制使得活性启动子和CFTR cDNA无法同时递送至靶细胞。我们推测，片段反式剪接（其中两个AAV载体分别递送CFTR cDNA的5'和3'半段）可将两个前体mRNA剪接成全长功能性CFTR mRNA。使用在第14a和14b外显子之间切割CFTR cDNA的片段反式剪接5'供体-3'受体对，将供体质粒和受体质粒共转染到CFTR（-）细胞中，可产生全长CFTR信息和蛋白质。将质粒显微注射到CFTR（-）细胞中可产生cAMP激活的氯离子传导。用工程化的人血清型AAV6.2构建的载体用于递送CFTR供体和受体构建体，在CFTR（-）细胞中产生全长CFTR mRNA和蛋白质以及cAMP激活的氯离子传导，包括人囊性纤维化气道上皮IB3-1细胞。因此，片段反式剪接可与AAV载体一起用于介导CFTR的表达，这一策略可能适用于囊性纤维化患者。
