New Perspectives on Antisense Oligos: The Long and the Short of it

In 1978, Zamecnik and Stephenson first demonstrated the ability to target and modulate gene expression through short synthetic oligonuceotides (Zamecnik and Stephenson, 1978, Zamecnik and Stephenson, 1978). Since then, antisense oligonucleotide (ASO)-based therapeutics have been studied extensively. However, only a single ASO drug has obtained FDA approval since their initial discovery in 1979 (Fomivirsen, Isis Therapeutics). As with many therapeutic strategies, target affinity, biostability, delivery and off-target effects have been central to the challenges facing ASO-based therapy in the clinic. To combat these obstacles, various aspects of the oligonucleotides have been altered, including the length of the oligonucleotide. Traditionally, the length of an ASO has been ~20-22 nucleotides, however recent evidence by Keil et al. and Pedersen et al. emphasizes the fact that shortening an ASO may be an effective targeting strategy in multiple contexts (Keil et al., 2014, Pedersen et al,. 2014).

Spinal Muscle Atrophy (SMA) is an autosomal recessive neurodegenerative disorder of motor neurons and the leading genetic cause of infant mortality. It is caused by a mutation that induces mis-splicing and the deletion of exon 7 in the SMN2 gene. An ASO can be used to control splicing by targeting a sequence in the pre-mRNA to prevent the interaction of various splicing modulators. For example, splicing silencers are sequences to which splicing repressor proteins bind, thereby decreasing the probability that a neighboring site will be used as a splice junction. Previously it was shown that a longer ASO can target intronic splicing silencer N1 (ISS-N1) and restore exon 7 to SMN2 (Singh et al., 2006), however a short ASO offers several advantages over their longer counterparts. These advantages include: a lower tolerance for mismatches, a reduction in the cost of synthesis, lower chance of stimulating the immune system, and increased probability of crossing biological barriers. However, the pervading thought has been that oligos this short will be cleared from the body too rapidly to be therapeutically effective and thus have not been extensively tested.

Previously, the authors performed an extensive antisense microwalk against ISS-N1 and the upstream and downstream regions (Singh et al., 2009). They found an 8-mer targeting a GC-rich region restored exon 7 inclusion in SMN2 at a moderate dose in a SMA patient derived cell line. Furthermore, this 2'OMe modified 8-mer showed zero tolerance for mismatch. In their most recent paper, they test efficacy in vivo in two different established mouse models for SMA. While the 2'O methylated 8-mer from their previous paper showed little effect in vivo, they found that stabilization of the ASO through the incorporation of a 5′ PEG-282 and a 3′ C3 propyl greatly improved the amount of SMN2 protein containing exon 7 in the liver. Furthermore, they show that the newly enhanced 8-mer improved weight gain and righting reflex and extended survival time in both mouse models. Though they found greater effects with the less severe mouse model, the authors note that their results closely mimic previous studies performed with long ASO.

Additional interesting research also considering optimal ASO length and affinity has recently been published (Pedersen et al., 2014). While advancement in nucleic acid chemistries has been essential in driving the success of shorter ASOs, it hasn't been appreciated until now how the reduction in length (thus affinity) directly affects the potency of an enzyme-recruiting ASO. In short, their research is the first to consider the kinetics of the binding of RNase-H to the complex and oligonucleotide. Their findings emphasizes the overall importance of the specific target (turnover rate) and cell type (RNase-H concentration) on optimal binding affinity and reconfirms that a shorter and less affine oligonuclotide can be more potent due to the faster rate of dissociation from a mismatched sequence. Taken together, these studies challenge the concept that a long ASO is superior to a short ASO in various contexts furthering current knowledge of ASO design strategy.

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