Cryptic U2-dependent pre-mRNA splice site usage 1 induced by splice switching antisense oligonucleotides 2

16 Antisense oligomers (AOs) are increasingly being used for modulating RNA splicing in live cells, 17 both for research and for therapeutic purposes. While the most common intended effect of these 18 AOs is to induce skipping of whole exons, rare examples are emerging of AOs that induce skipping 19 of only part of an exon, through activation of an internal cryptic splice site. In this report, we 20 examined seven such examples of AO-induced cryptic splice site activation – five new examples 21 from our own experiments and three from reports published by others. We modelled the predicted 22 effects that AO binding would have on the secondary structure of each of the RNA targets, and 23 how these alterations would in turn affect the accessibility of the RNA to splice factors. We observed that a common predicted effect of AO binding was a disruption to the exon definition 25 signal within the exon’s excluded segment. S.F. the of Western to Sarepta Therapeutics and as are entitled to and payments; K.A.H., C.S.M., M.T.A-H., K.G. support from Sarepta Therapeutics. The funders no role in the design of the study; in the collection, analyses, or interpretation of data; in the of the or in the decision to publish the results. N.P.K and K.Z declare no competing interests.


Introduction 27
The process of pre-mRNA splicing is a fundamental aspect of gene regulation and function in higher

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Alternative splicing is a process whereby multiple different transcript and protein isoforms can 46 arise from a single protein-coding gene and is an essential element in spatial and temporal regulation 47 of gene expression in higher eukaryotes 7 . In order to achieve alternative splicing, the spliceosome 48 must recognize and select a splice site from a variety of alternative splice sites and branchpoints 49 within the transcript. Typically, these splice sites are well defined and have evolutionarily conserved 50 functions. However, sometimes sequences usually ignored by the spliceosome can become 51 2 activated as splice junctions. These are known as cryptic splice sites 11 and are most often activated 52 by mutations or errors during transcription 12 . The most common causative mutations are those that 53 abolish canonical splice sites, thus redirecting the spliceosome to either utilize a viable cryptic site 54 nearby or exclude the exon completely from the mature mRNA 13 . Cryptic splice sites may be found 55 within both exonic and intronic regions and typically include or exclude a proportion of the intron or 56 exon 12 . Interestingly, recent data has shown that cryptic splice sites can also be activated by 57 synthetic molecules such as antisense oligonucleotides.

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Antisense oligonucleotides (AOs) are small, single-stranded RNA or DNA-like synthetic 59 molecules used to modify gene expression. These AOs can be used to downregulate gene 60 expression through RNA silencing, redirection of pre-mRNA splicing patterns, intron retention, 61 inhibiting translation, or RNase H-induced degradation of the target gene transcript 14 . The sequence 62 of maturing gene transcripts can also be altered by using AOs to induce removal or inclusion of an 63 exon, as seen with current therapeutic strategies approved for the treatment of Duchenne muscular 64 dystrophy and spinal muscular atrophy, respectively.

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While most splice modulating AOs are designed with the intention to enhance exon selection 66 or induce skipping of whole exons, the occasional activation of cryptic splice sites after in vitro AO 67 treatment has also been observed. We have reported the activation of a cryptic donor splice site 68 after treatment with an AO targeting LMNA pre-mRNA, promoting removal of 150 nt from the end of 69 exon 11 15 . Evers et al. 16 observed that an AO targeting exon 9 in ATXN3 promoted a partial exon 9 70 skip, activating an alternative 5'ss. A partial exon 12 skip in the HTT transcript was also detected 71 after treatment with an AO (World Patent WO2015053624A2); once again activating a cryptic donor 72 splice site 17 . Lastly, we recently reported activation of two cryptic donor splice sites by AOs 73 containing several locked nucleic acid residues, designed to enhance efficiency of exon skipping 74 from the dystrophin transcript 18 .

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In addition to the established roles that splice site motifs and exon enhancer and silencer motifs 76 play in directing RNA splicing, there is increasing evidence of a similar role for RNA secondary 77 structure 19-22 and of its effect on splice factor binding 23,24 . While modelling the interactions of these 78 phenomena presents a highly complex challenge, a reasonable starting point may be to assume that 79 RNA secondary structure is generally antagonistic to splice factor binding within closed regions.

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In our laboratory's quest to develop new therapeutics for debilitating genetic diseases, we have 81 tested thousands of AOs targeted to numerous genes in a variety of cell types, but we have observed 82 only a handful of AO-induced cryptic splicing events in the target transcripts in human cells, and a 83 single example in mouse cells 25 . In this study, we investigated the possible mechanisms by which 84 AOs may induce cryptic splicing. We analyzed 13 AOs targeting six different human gene transcripts 85 and found that changes to the accessibility of enhancer and silencer motifs within the transcript 86 secondary structure appeared to play a role in many cases. The diverse nature of these changes 87 indicates that there may be multiple pathways to inducing cryptic splicing, sometimes within a single 88 exon.

Results and Discussion 91
To explore the possible mechanisms behind cryptic splice site activation, we analyzed AO-induced     Fig. 1a). The splicing of T202 appears to be influenced by 120 the AOs in the same manner ( Supplementary Fig. 1a). However, we were unable to isolate and 121 identify various amplicons to confirm this. The AOs did not appear to cause exon skipping or cryptic 122 donor site activation within the T203 transcript, most likely due to the T203 isoform containing only 123 two exons, making both "unskippable" 26 .

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Under normal conditions, SRSF2 transcript isoforms T202 and T203 code for proteins while 125 T208 and T204 undergo nonsense mediated decay (NMD). After AO treatment, the expression of the 126 cryptically spliced T208 increased with a concomitant decrease in the full-length T202. The cryptic 127 splicing of exon 2 removes the natural termination codon from T202, T204, and T208 and exposes 128 a new in-frame termination codon in the following exon of each transcript ( Supplementary Fig. 1b).

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Mammalian NMD generally follows the '50 nucleotide rule', whereby termination codons more 130 than 50 nt upstream of the final exon are determined premature and result in a reduction in mRNA 131 abundance 27 . Cryptic splice site activation appears to stabilize T208 as a new termination codon is 132 created within 50 nt of the penultimate 3' exon junction. Isoform T204 still appears to undergo NMD, 133 as the new termination codon is exposed within the third exon of the five-exon isoform.

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The same AO treatment also activated a cryptic donor site, resulting in removal of 55 nt from the 8Q 143 transcript. Treatment with H9D(+20-05) resulted solely in partial exon 9 skipping from the 8Q 144 transcript. All amplicons were isolated and identified by Sanger sequencing.

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Complete and partial exon 9 skipping was observed only from the 8Q and not the 21Q    (Table   187 1), indicating splice site scores are not the only factor influencing splice site usage. 197

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The splicing factor SRSF1 is necessary for several splicing processes, including lariat formation

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In COL7A1 exon 15 (Fig. 2a), AO binding was predicted to increase ESE access in the retained 230 5′ segment, as well as directly competing with ESEs in the excised 3′ segment. The net effect was 231 a much stronger exon signal from the 5′ segment that improved the profile of the cryptic donor site.

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For SRSF2 exon 2 (Fig. 2b), the AO directly obscured the strongest enhancer peak in the 236 excised 3' segment and induced a moderate increase in ESE access within the retained 5' segment.

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We also observed that, in the absence of AO binding, the enhancer signal in the excised 3' segment

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In HTT exon 12 (Fig. 2d), the changes in secondary structure did not clearly favor either 246 enhancement or silencing of the excised segment. However, ESS access was increased both 5' and 247 3' of the canonical donor site, and this appears to have been sufficient to tip the balance towards 248 the comparably strong cryptic donor splice site. A similar change appears to have occurred in LMNA 249 exon 11 (Fig. 2e), with the exception that the cryptic donor site in this exon was much stronger than 250 its canonical neighbor.

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For USH2A exon 13, there was almost no change to predicted secondary structure induced by 252 H13A(+70+94), apart from that at the AO binding site (Fig. 2f). It therefore appears that steric blocking   Table 3.

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For example, a nucleotide that fell within two six nt ESE motifs and one eight nt ESS motif would 361 be assigned an ESE score of 0.333 (2 x 1/6) and an ESS score of -0.125 (1 x -1/8).

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Predicted centroid normal RNA folding was calculated for the sequence of each cryptically 364 spliced exon with +/-70 nt flanking intron, using RNAfold 43 with the "avoid isolated base pairs" 365 option. Predicted centroid AO-induced folding was calculated for each exon using the same 366 sequence and settings as for normal folding, but with an additional constraint mask that prohibited 367 binding within the AO target sites.

Data availability 370
All data generated or analyzed during this study are included in this published article (and its