Modulating the expression of disease genes with RNA-based therapy

Abstract

Conventional gene therapy has focused largely on gene replacement in target cells. However, progress from basic research to the clinic has been slow for reasons relating principally to the challenges of heterologous DNA delivery and regulation in vivo. Alternative approaches targeting RNA have the potential to circumvent some of these difficulties, particularly as the active therapeutic molecules are usually short oligonucleotides and the target gene transcript is under endogenous regulation. RNA-based strategies offer a series of novel therapeutic applications, including altered processing of the target pre-mRNA transcript, reprogramming of genetic defects through mRNA repair, and the targeted silencing of allele- or isoform-specific gene transcripts. This review examines the potential of RNA therapeutics, focusing on antisense oligonucleotide modification of pre-mRNA splicing, methods for pre-mRNA trans-splicing, and the isoform- and allele-specific applications of RNA interference.

Figure 1. AO-Based Manipulation of Pre-mRNA Splicing

(A) Blockage of cryptic splicing as a therapy for β-thalassemia. Mutations within intron 2 of the β-globin gene induce usage of cryptic splice sites that incorporate intronic sequence into the mature mRNA. Disruption of the reading frame introduces a stop codon that results in truncated β-globin protein. Blockage of the cryptic 5′ splice site with AO (blue bar) restores normal splicing pattern and functional β-globin protein is produced. (B) Restoration of dystrophin production in the mouse model of DMD by exon exclusion. A C→T mutation in exon 23 of the mouse dystrophin gene introduces a stop codon that produces a truncated nonfunctional protein. Blockage of the 5′ splice site of exon 23 disrupts its recognition by splicing machinery, resulting in removal of the in-frame exon from the dystrophin transcript. This facilitates translation of near full-length, semi-functional dystrophin protein. (C) Exon inclusion to increase production of SMN protein as a therapy for SMA. A silent mutation in the SMN2 gene disrupts an ESE site in exon 7, preventing binding of the SF2/ASF splicing factor and affecting exon recognition such that the majority of SMN2 transcripts lack exon 7, producing a poorly functional SMN protein. Targeting with a bifunctional AO (blue bar) containing a functional ESE sequence recruits the SF2/ASF factor, promoting exon recognition and incorporation into the mature transcript, resulting in translation of normal SMN protein.

Figure 2. RNA Trans-Splicing

(A) Correction of CF mutations in the CFTR gene using SMaRT. A PTM containing a binding domain (BD), splicing domain (black line), and a coding domain (orange) incorporating exons 10–24 of wild-type CFTR mRNA, binds to intron 9 of CFTR pre-mRNA (green) containing disease-causing mutations (stars). SMaRT removes the mutant pre-mRNA such that reprogrammed transcript containing wild-type mRNA allows synthesis of a functional protein. (B) Ribozyme-mediated trans-splicing for application to trinucleotide repeat expansions. Large (50–2,000) CUG repeat expansion in the 3′ untranslated region of the DMPK gene cause myotonic dystrophy. Ribozymes containing a reduced number of CUG repeats are targeted to the mutant DMPK transcript (green) via complementary binding mediated by a guide sequence (black bars). Binding of the ribozyme facilitates cleavage of the DMPK mRNA and trans-splicing of the coding region (orange) and smaller CTG repeat expansion to produce a non-toxic DMPK mRNA transcript.

Figure 3. RNAi for Isoform- and Allele-Specific Silencing

(A) Isoform-specific RNAi to target disease-associated isoforms in cancer. VEGF₁₆₅ isoform overexpression is implicated in tumour angiogenesis. Targeting of the VEGF transcript with siRNA targeted to the exon 5/7 boundaries, in association with RISC, induces specific VEGF₁₆₅ knockdown, while having no effect on other VEGF isoforms, e.g., VEGF₁₈₉. (B) Allele-specific RNAi in the autosomal dominant slow channel congenital myasthenic syndrome. A missense mutation (red bar) in the muscle acetylcholine α-subunit (αS226F) induces a C→U change in the mutant allele. Use of siRNA specific for the αS226F mutation (A binding to U), induces discriminatory silencing of the mutant transcript, leaving the wild-type transcript mostly unaffected.