Development of Therapeutic Splice-Switching Oligonucleotides

Abstract

Synthetic splice-switching oligonucleotides (SSOs) target nuclear pre-mRNA molecules to change exon splicing and generate an alternative protein isoform. Clinical trials with two competitive SSO drugs are underway to treat Duchenne muscular dystrophy (DMD). Beyond DMD, many additional therapeutic applications are possible, with some in phase 1 clinical trials or advanced preclinical evaluation. Here, we present an overview of the central factors involved in developing therapeutic SSOs for the treatment of diseases. The selection of susceptible pre-mRNA target sequences, as well as the design and chemical modification of SSOs to increase SSO stability and effectiveness, are key initial considerations. Identification of effective SSO target sequences is still largely empirical and published guidelines are not a universal guarantee for success. Specifically, exon-targeted SSOs, which are successful in modifying dystrophin splicing, can be ineffective for splice-switching in other contexts. Chemical modifications, importantly, are associated with certain characteristic toxicities, which need to be addressed as target diseases require chronic treatment with SSOs. Moreover, SSO delivery in adequate quantities to the nucleus of target cells without toxicity can prove difficult. Last, the means by which these SSOs are administered needs to be acceptable to the patient. Engineering an efficient therapeutic SSO, therefore, necessarily entails a compromise between desirable qualities and effectiveness. Here, we describe how the application of optimal solutions may differ from case to case.

Systemic peptide-mediated oligonucleotide therapy improves long-term survival in spinal muscular atrophy

“…Here, we report that a highly active peptide, Pip6a, directly conjugated to a morpholino PMO permits highly efficient systemic delivery, which enhances bodywide SMN expression, including in brain and spinal cord; rescues the phenotype; and dramatically prolongs the life span of severe SMA mice. These data demonstrate powerful SMA disease modification by peptide-PMO therapy, a benefit that could be extended to many other neurodegenerative disorders (8,31,32). …”

RNA therapeutics inactivate PCSK9 by inducing a unique intracellular retention form

“…The 2′-O-methyl-phosphorothioate chemistry used in this study is one of the oldest and best characterized in the field of oligonucleotides -mediated gene therapy in terms of specificity, efficiency, toxicity and bio-distribution and is currently being employed in clinical trials [43]. One major characteristic of oligonucleotides with this chemistry (as for many other SSOs chemistries) is their preferential accumulation in the liver and kidney [44], making them excellent therapeutics for liver-related diseases.…”

Antisense Oligonucleotide Therapy for Inherited Retinal Dystrophies

“…This process is carried out by the spliceosome, and involves multiple interactions mediated by splicing factors that recognize regulatory elements in the target pre-mRNA molecules (Hastings and Krainer 2001). It is estimated that up to 50 % of disease-causing mutations affect pre-mRNA splicing (Disterer et al 2014), with obvious consequences at the protein level. Thus, altering splicing offers an interesting therapeutic strategy for many genetic disorders (Hammond and Wood 2011).…”

“…Subsequently, different generations of oligonucleotides have been developed, with chemical modifications to make them resistant to the RNase-H activity, increase their half-life and improve their binding affinity (Chan et al 2006). A major class of AONs are those with a phosphorothioate backbone, and based on the chemistry 2ʹ-O-methyl or 2ʹ-O-methoxyethyl, or the phosphoramidate morpholino oligonucleotides (Chan et al 2006;Disterer et al 2014). These molecules have a high ability to interfere with splicing, either by masking splice sites, or by targeting regulatory sequences to promote or block splicing (Hammond and Wood 2011).…”