Antisense-Mediated RNA Targeting: Versatile and Expedient Genetic Manipulation in the Brain

Abstract

A limiting factor in brain research still is the difficulty to evaluate in vivo the role of the increasing number of proteins implicated in neuronal processes. We discuss here the potential of antisense-mediated RNA targeting approaches. We mainly focus on those that manipulate splicing (exon skipping and exon inclusion), but will also briefly discuss mRNA targeting. Classic knockdown of expression by mRNA targeting is only one possible application of antisense oligonucleotides (AON) in the control of gene function. Exon skipping and inclusion are based on the interference of AONs with splicing of pre-mRNAs. These are powerful, specific and particularly versatile techniques, which can be used to circumvent pathogenic mutations, shift splice variant expression, knock down proteins, or to create molecular models using in-frame deletions. Pre-mRNA targeting is currently used both as a research tool, e.g., in models for motor neuron disease, and in clinical trials for Duchenne muscular dystrophy and amyotrophic lateral sclerosis. AONs are particularly promising in relation to brain research, as the modified AONs are taken up extremely fast in neurons and glial cells with a long residence, and without the need for viral vectors or other delivery tools, once inside the blood brain barrier. In this review we cover (1). The principles of antisense-mediated techniques, chemistry, and efficacy. (2) The pros and cons of AON approaches in the brain compared to other techniques of interfering with gene function, such as transgenesis and short hairpin RNAs, in terms of specificity of the manipulation, spatial, and temporal control over gene expression, toxicity, and delivery issues. (3) The potential applications for Neuroscience. We conclude that there is good evidence from animal studies that the central nervous system can be successfully targeted, but the potential of the diverse AON-based approaches appears to be under-recognized.

Peripheral Administration of Antisense Oligonucleotides Targeting the Amyloid-β Protein Precursor Reverses AβPP and LRP-1 Overexpression in the Aged SAMP8 Mouse Brain

“…Given that the BBB is also a dynamic regulatory interface for bidirectional peptide transport [2,3], there is much interest in utilizing these transport systems for drug delivery to the brain. Previously, our lab and others have used phosphorothioated antisense oligonucleotides to modify protein expression in vivo [4][5][6][7]. We have also demonstrated that these antisense oligonucleotides have a saturable BBB transport system which allows them to access the CNS and alter expression of their protein targets [5,8,9].…”

Antisense Oligonucleotide Mediated Splice Correction of a Deep Intronic Mutation in OPA1

“…Moreover, AONs are small molecules that easily penetrate into tissues and are taken up by neurons without complex chemical formulations or packaging vehicles. 30 For its application to treat optic atrophy and thus targeting retinal ganglion cells an administration through intravitreal injection can be envisaged. Another advantage of AON over classical gene therapy approaches is the possibility to discontinue treatment at any time, in case of side-effects which may occur.…”

Ataxin-3 Protein and RNA Toxicity in Spinocerebellar Ataxia Type 3: Current Insights and Emerging Therapeutic Strategies

“…With exon skipping, AONs are used that target-specific splicing signals, masking an exon in the pre-mRNA from the splicing machinery and thus resulting in exclusion of the target exon from the mRNA [208, 209]. When the correct reading frame is maintained, subsequent translation will result in formation of an internally truncated protein.…”