Functional repair of a mutant chloride channel using a trans-splicing ribozyme

Abstract

IntroductionAdvances in molecular genetics have fostered the development of new therapeutic approaches, such as genebased therapy, for the treatment of inherited disorders. The most widely used strategy involves the introduction of wild-type sequence into cells with a defective gene. This strategy is best suited for the treatment of inherited diseases in which there is complete absence of functional alleles, such as in recessive disorders. However, it is not clear that this approach will succeed in all disorders, especially those with a dominant-negative mechanism of disease pathogenesis.Strategies using repair of mRNA are alternative approaches that will conceptually work in both dominant and recessive disorders (1). Replacing the defective portion of an mRNA molecule reduces the fraction of mutant transcripts and increases the absolute level of the normal (or repaired) transcript. The advantages of this strategy are that the target mRNA remains under the control of its endogenous promoter, thereby assuring proper regulation of expression, and tissue specificity is achieved by targeting mRNA only in cells expressing the mutant gene. RNA repair has been accomplished by two similar methods: spliceosome-mediated (2) and ribozyme-mediated trans-splicing (3-6).The Tetrahymena group I intron ribozyme has been modified to facilitate trans-splicing of its 3′ exon onto separate RNA molecules in a sequence-specific manner (7,8). This trans-splicing reaction is initiated by complementary base pairing of the target RNA to the ribozyme internal guide sequence (IGS). The ribozyme then catalyzes the cleavage of the target RNA immediately downstream of the IGS binding site and the subsequent ligation of its 3′ exon to the remaining target RNA, generating a complete repaired RNA transcript. Ribozyme-mediated transsplicing has been used to repair defective β-globin (9), myotonic dystrophy protein kinase (10), and p53 transcripts (11) in cultured mammalian cells. To date, the longest trans-spliced 3′ exon has been 1.1 kb (11), but the ability of the Tetrahymena ribozyme to mediate trans-splicing of larger exons has not been determined. Furthermore, the efficiency of the trans-splicing reaction has not been accurately measured in a cell-based system. Importantly, repaired transcripts must be translated into functional protein, but only one study has demonstrated restored protein function determined indirectly in lysates from a population of cells (11).To further investigate the feasibility of RNA repair, we designed and tested the ability of a modified Tetrahymena ribozyme to mediate trans-splicing repair of a mutant canine skeletal muscle chloride channel (cClC-1) mRNA transcript that causes the inherited disorder myotonia congenita. We demonstrated that this ribozyme can mediate trans-splicing of a large (4 kb) 3′ exon in cultured cells. Furthermore, we measured trans-splicing reaction

The non-dystrophic myotonias: molecular pathogenesis, diagnosis and treatment

“…Such a technique has been employed using a transsplicing ribozyme to restore a mutant chloride channel transcript in a cellular model. Although a good recovery of chloride channel function was observed in individual cells the efficiency of RNA repair in the cell culture as a whole was only 1.2% (Rogers et al, 2002).…”

Treatment of Neuromuscular Channelopathies: Current Concepts and Future Prospects

“…This technique has been used on a canine mutant chloride channel mRNA transcript, and despite a low level of repair efficiency (approximately 1%), up to 18% of cells were found to have detectable chloride currents. 22 Whether or not this technique can be applied to an in vivo animal model of the disease, and thence to humans, remains to be seen. Furthermore, whether oral AON therapy is possible in channel disorders also remains to be seen.…”

Épissage alternatif, pathologie et thérapeutique moléculaire

“…Elle a permis la correction du saignement, associé à la production de facteur VIII, dans un modèle de souris hémophiles [41]. Le trans-épissage a été également utilisé pour cibler des transcrits matures avec des ribozymes de groupe I [42]. Une troisième approche repose sur l'utilisation d'une endonucléase d'épissage des ARNt.…”