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 5' splice site usage, alternate 3' 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 of ISS-N1-like sequences in the
affected exons and/or their flanking introns. Our results will reveal the required minimum complementarity
between ASO and its target for the antisense response. We will determine if the chemistry-specific off-target
effect of an N1ASO is due to a common motif within the target sequence. Outcomes of our proposed study will
have high significance for developing future ASO-based therapies for SMA as well as other diseases amenable
by ASOs encompassing PS/OMe, PS/MOE and morpholino modifications.