Transcriptome-Wide Effect of Therapeutic Antisense Oligonucleotides for the Treatment of Spinal Muscular Atrophy - Spinal muscular atrophy (SMA) is one of the leading genetic causes of infant mortality. SMA is caused by deletions of or mutations in the Survival Motor Neuron 1 (SMN1) gene that codes for the multifunctional SMN protein. SMN2, a nearly identical copy of SMN1, fails to fully compensate for the loss of SMN1 due to predominant skipping of exon 7. Prevention of SMN2 exon 7 skipping is a proven approach for the treatment of SMA. Among many regulatory elements that modulate SMN2 exon 7 splicing, intronic splicing silencer N1 (ISS-N1) has been extensively validated as an antisense oligonucleotide (ASO)-directed therapy for SMA. Nusinersen, an ISS-N1-targeting ASO (abbreviated as “N1ASO”), became the first FDA-approved drug for SMA. Risdiplam, a small molecule capable of preventing SMN2 exon 7 skipping, is also approved for SMA therapy. Gene replacement is an additional therapeutic avenue for the treatment of SMA. Current therapies of SMA do not fully meet the needs of SMA patients, as most severe SMA patients remain wheelchair-bound even after several years of treatment. One of the likely causes of low efficacy of available drugs for SMA is the off-target effects at concentrations used for clinical applications. Phosphorothioate (PS) backbones present within ASOs are known to produce off-target effects. However, off-target effects of 2¢O-Methyl (OMe) or 2¢O- Methoxyethyl (MOE) incorporations in the sugar moiety of RNA oligonucleotides remain largely unknown. Additional off-target effects of ASOs could be expected due to tolerance for mismatch base pairing with the target. High concentration of a 20mer N1ASO encompassing PS/OMe modifications has been recently reported to cause massive perturbations of the transcriptome in SMA patient cells. However, similar study has not yet been done with Nusinersen, an 18mer N1ASO encompassing PS/MOE modifications. Also, there is no comparative study on the concentration-dependent chemistry-specific off-target effects of N1ASOs. Unlike PS/OMe and PS/MOE ASOs that contain negatively charged backbones, morpholino ASOs have neutral backbones. We performed RNA-Seq on transcripts generated from SMA patient cells treated with N1ASOs encompassing PS/OMe, PS/MOE and morpholino modifications. Our preliminary analysis and pilot validations of RNA-Seq data reveal chemistry-specific perturbations of the transcriptome. Here we propose to fully analyze and validate the results of RNA-Seq. In Aim 1, we will determine the nature of genes and pathways impacted by an N1ASO encompassing three chemistries. We will determine the extent to which small, large, coding, non-coding genes are affected by the chemistry of an N1ASO. We will validate upregulated and downregulated genes as well as seven types of aberrant splicing events, including exon skipping, exon inclusion, enhanced intron retention, enhanced intron removal, alternate 5ʹ splice site usage, alternate 3ʹ splice site usage and mixed splicing events. We will analyze promoters and upstream sequences of upregulated and downregulated genes to determine the presence of common motifs responsive to N1ASOs. We will examine if the off-target effect on splicing is associated with splice site strengths of the affected exons. We will determine if ISS-N1-like sequences within the affected exons and/or their flanking introns are associated with the off-target splicing. We will examine if transfection conditions also affect the nature of off-target effects of N1ASOs. We will determine if some of the off-target effects of N1ASOs are cell-type specific. In Aim 2, we will determine primary and secondary targets of N1ASOs by capturing the antisense effects at early and late timepoints of the treatment with N1ASOs, respectively. We will examine both sequence-dependent and sequence-independent targets. We will employ minigenes to uncover the nature of sequences associated with the aberrant splicing triggered by N1ASOs encompassing different chemistries. We will interrogate the role
