mRNAtrans‐splicing in gene therapy for genetic diseases

Abstract

Spliceosome‐mediated RNA trans‐splicing, or SMaRT, is a promising strategy to design innovative gene therapy solutions for currently intractable genetic diseases. SMaRT relies on the correction of mutations at the post‐transcriptional level by modifying the mRNA sequence. To achieve this, an exogenous RNA is introduced into the target cell, usually by means of gene transfer, to induce a splice event in trans between the exogenous RNA and the target endogenous pre‐mRNA. This produces a chimeric mRNA composed partly of exons of the latter, and partly of exons of the former, encoding a sequence free of mutations. The principal challenge of SMaRT technology is to achieve a reaction as complete as possible, i.e., resulting in 100% repairing of the endogenous mRNA target. The proof of concept of SMaRT feasibility has already been established in several models of genetic diseases caused by recessive mutations. In such cases, in fact, the repair of only a portion of the mutant mRNA pool may be sufficient to obtain a significant therapeutic effect. However in the case of dominant mutations, the target cell must be freed from the majority of mutant mRNA copies, requiring a highly efficient trans‐splicing reaction. This likely explains why only a few examples of SMaRT approaches targeting dominant mutations are reported in the literature. In this review, we explain in details the mechanism of trans‐splicing, review the different strategies that are under evaluation to lead to efficient trans‐splicing, and discuss the advantages and limitations of SMaRT. WIREs RNA 2016, 7:487–498. doi: 10.1002/wrna.1347For further resources related to this article, please visit the WIREs website.

Can Adeno-Associated Viral Vectors Deliver Effectively Large Genes?

“…Although initially trans-splicing has been explored to correct mutations in endogenously produced mutated pre-mRNAs by delivery of an exogenous corrected fragment of the endogenous gene, 82 adaptations of this process have been later developed. In the approach defined as ''segmental trans-splicing'' (STS), two engineered individual DNA fragments encode for the 5¢ and 3¢ fragments of the pre-mRNA of a large gene and share an intronic hybridization domain that can favor trans-splicing, leading to joining of the two half-transcripts into an intact full-length mRNA 83 (Fig.…”

“…Interestingly, AAV-mediated delivery of the 5¢-and 3¢-halves of the CFTR cDNA in human CF airway epithelial cells followed by STS was found to result in full-length CFTR reconstitution and restoration of CF cells' functionality. 84 Despite this pivotal proof-of-concept study, however, the inability to achieve significant levels of pre-mRNA trans-splicing, as well as the several scientific and technical challenges related to the use of RNA-based repair strategies 82,85 have limited further investigation of STS in combination with AAV vectors as a strategy for corrections of mutations in large genes.…”

High‐Throughput Assays to Assess the Functional Impact of Genetic Variants: A Road Towards Genomic‐Driven Medicine

“…The exogenous molecule contains the desirable sequence to replace the aberration, resulting in a chimeric transcript that encodes for a functional protein. Despite poor in vivo efficacy of the trans‐splicing process, therapies towards treating diseases such as cystic fibrosis, spinal muscular atrophy, Duchenne muscular atrophy, and retinitis pigmentosa are being tested.…”

Targeting Splicing in the Treatment of Human Disease

“…Designing a correct PTM is crucial for the reprogramming of mRNA. The PTM must carry the wild-type coding region of the gene that is to be replaced, 5′ and 3′ SS, intronic BPS and Py sequences, and a complementary sequence or binding domain for precise and specific hybridization to the mutated pre-mRNA (Figure 4, panel b) [62,63]. …”

“…Recently, the trans -splicing system has been optimized through the combination of the trans-splicing RNA and antisense RNA interfering with competitive splicing elements on the pre-mRNA [70,71]. A comprehensive review detailing the use of SMaRT in gene therapy for genetic diseases has been recently published [63]. …”