Modulating the Expression of Disease Genes with RNA-Based Therapy

onventional 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. Antisense Oligonucleotide-Based Manipulation of Pre-mRNA SplicingTraditionally, antisense oligonucleotides (AOs) have been employed to down-regulate gene transcription, through either RNase-H mediated degradation or steric blockage of gene promoter elements.More recently however, the potential of using AOs to alter pre-mRNA processing is being realised.Through utilisation of AO chemistries that do not induce transcript degradation, targeted blockage of motifs involved in splicing allows the manipulation of this process.Given that in the Human Gene Mutation Database (http:// www.hgmd.cf.ac.uk/ac/index.php);10% of annotated mutations impinge on splice sites [1], there is the potential for this approach to be applied to diseases caused by aberrant splicing, or where alteration of normal splicing would abrogate the disease-causing mutation.This could include: (i) blockage of cryptic splice sites, (ii) exon removal or inclusion to alter isoform expression, and (iii) removal of exons to either eliminate a nonsense mutation or restore the reading frame around a genomic deletion.Blockage of cryptic splicing.The concept of using AOs to alter pre-mRNA splicing was first demonstrated in bthalassemia, which is caused by mutations in the human bglobin gene [2].The most prevalent disease-causing mutations disrupt splicing of introns 1 and 2 of the b-globin pre-mRNA transcript through activation of cryptic splice sites, preventing production and translation of the correct mRNA [3].Blockage of these cryptic splice sites with AOs in erythroid cells abrogates their use, restoring normal globin expression [4] (Figure 1A).Similarly, in Hutchinson-Gilford progeria syndrome, a point mutation in exon 11 of the lamin A/C (LMNA) gene causes a silent substitution (GGCfiGGT) that results in activation of a cryptic donor splice site [5].Splicing between this cryptic splice site and the acceptor splice site gives rise to a truncated LMNA mRNA containing a