activation of SMN by selective disruption of PNAS PLUS lncRNA-mediated recruitment of PRC2 for the treatment of spinal muscular atrophy

Caroline J. Wooa,1, Verena K. Maiera, Roshni Daveya, James Brennana, Guangde Lia, John Brothers IIa, Brian Schwartza, Susana Gordoa, Anne Kaspera, Trevor R. Okamotob, Hans E. Johanssonb, Berhan Mandefroc,d, Dhruv Sareenc,d,e, Peter Bialeka, B. Nelson Chaua, Balkrishen Bhata, David Bullougha, and James Barsouma

aRaNA Therapeutics, Cambridge, MA 02139; bLGC Biosearch Technologies, Petaluma, CA 94954; cBoard of Governors–Regenerative Medicine Institute, Cedars–Sinai Medical Center, Los Angeles, CA 90048; dInduced Pluripotent Stem Cell Core, The David and Janet Polak Stem Cell Laboratory, Cedars–Sinai Medical Center, Los Angeles, CA 90048; and eDepartment of Biomedical Sciences, Cedars–Sinai Medical Center, Los Angeles, CA 90048

Edited by Robert E. Kingston, Massachusetts General Hospital/Harvard Medical School, Boston, MA, and approved January 10, 2017 (received for review October 4, 2016) Spinal muscular atrophy (SMA) is a neurodegenerative disease predict that modest SMN2 up-regulation will provide significant characterized by progressive motor neuron loss and caused by therapeutic benefit. Here, we establish that PRC2 interacts with a mutations in SMN1 (Survival Motor Neuron 1). The disease severity long noncoding RNA (lncRNA) transcribed within the SMN2 inversely correlates with the copy number of SMN2, a duplicated locus and regulates SMN2 expression through PRC2-associated gene that is nearly identical to SMN1. We have delineated a mech- epigenetic modulation. Furthermore, we demonstrate that we can anism of transcriptional regulation in the SMN2 locus. A previously selectively up-regulate SMN2 expression by interrupting the uncharacterized long noncoding RNA (lncRNA), SMN-antisense 1 lncRNA-mediated recruitment of PRC2 to the SMN2 locus. Such (SMN-AS1), represses SMN2 expression by recruiting the Polycomb an approach represents a therapeutic strategy for SMA and po- Repressive Complex 2 (PRC2) to its locus. Chemically modified oligo- tentially can be used to elevate the expression of target in nucleotides that disrupt the interaction between SMN-AS1 and PRC2 various human disease settings. inhibit the recruitment of PRC2 and increase SMN2 expression in pri- MEDICAL SCIENCES mary neuronal cultures. Our approach comprises a gene-up-regulation Results technology that leverages interactions between lncRNA and PRC2. PRC2 Modulates SMN2 Expression. Analysis of publicly available Our data provide proof-of-concept that this technology can be used chromatin immunoprecipitation (ChIP) sequencing data from the to treat disease caused by epigenetic silencing of specific loci. ENCODE consortium and the Broad Institute (genome.ucsc.edu/) (14, 15) suggests that PRC2 is associated with SMN2 in different spinal muscular atrophy | lncRNA | PRC2 | SMN cell types to varying degrees, most notably with the HepG2 cells (Fig. 1A). In addition, ChIP sequencing data from the NIH pinal muscular atrophy is the leading genetic cause of infant Roadmap Epigenome Consortium (16) suggests that H3K27me3, Smortality and is caused by deletions or mutation of Survival the hallmark of PRC2 activity, is associated with SMN2 in human Motor Neuron 1 (SMN1)(1).Uniquetohumans,SMN1 is dupli- fetal brain (SI Appendix,Fig.S1). To determine whether disruption cated in the genome as SMN2, which is nearly identical in se- SMN2 quence. However, a C-to-T point mutation in exon 7 of Significance results in preferential skipping of this exon during pre-mRNA splicing and production of a truncated and unstable . A Autosomal recessive mutations or deletions of the gene Survival small fraction (10–20%) of pre-mRNA transcribed from SMN2 Motor Neuron 1 (SMN1) cause spinal muscular atrophy, a neuro- is spliced correctly to include exon 7 and produces a full-length degenerative disorder. Transcriptional up-regulation of a nearly SMN (SMN-FL, inclusive of exon 7) that is identical to the identical gene, SMN2, can functionally compensate for the loss of SMN1 gene product (2–4). SMN1, resulting in increased SMN protein to ameliorate the dis- Spinal motor neurons are highly sensitive to SMN1 deficiency, ease severity. Here we demonstrate that the repressed state of and their premature death causes motor function deficit in SMA SMN2 is reversible by interrupting the recruitment of a repressive patients (5, 6). The SMN2-derived SMN protein can extend spinal epigenetic complex in disease-relevant cell types. Using chemically motor neuron survival, yet insufficient levels of SMN eventually modified oligonucleotides to bind at a site of interaction on a long lead to cell death. Overall, SMA patients with higher SMN2 ge- noncoding RNA that recruits the repressive complex, SMN2 is nomic copy number have a less severe disease phenotype (7, 8). epigenetically altered to create a transcriptionally permissive state. Type 0 or I patients, carrying one or two copies of SMN2, show

onset of SMA within a few months of life with a life expectancy of Author contributions: C.J.W., B.S., S.G., H.E.J., D.S., P.B., B.N.C., D.B., and J. Barsoum de- less than 2. In contrast, type III and IV patients, carrying three or signed research; C.J.W., V.K.M., R.D., J. Brennan, G.L., B.S., S.G., A.K., T.R.O., H.E.J., B.M., more copies, respectively, show juvenile or adult onset and slower and P.B. performed research; D.S., B.N.C., and B.B. contributed new reagents/analytic disease progression (9). As further genetic evidence, SMA mouse tools; C.J.W., V.K.M., R.D., J. Brennan, J. Brothers, B.S., S.G., A.K., T.R.O., H.E.J., P.B., and − − B.N.C. analyzed data; and C.J.W. wrote the paper. models have been produced in which smn1 / mice, which would Conflict of interest statement: C.J.W., V.K.M., R.D., J. Brennan, G.L., J. Brothers, B.S., S.G., otherwise be embryonic lethal (10), can be rescued in the presence A.K., P.B., B.N.C., B.B., D.B., and J. Barsoum declare a financial interest in the body of work of high copy numbers of the human SMN transgene (11–13). generated as shareholders and employees of RaNA Therapeutics. Similar to the human disease spectrum, increased copy number of This article is a PNAS Direct Submission. SMN ahuman transgene is inversely associated with decreased Freely available online through the PNAS open access option. SMN2 disease severity and mortality. We reasoned that increasing Data deposition: The data reported in this paper have been deposited in the Gene Ex- transcription could phenocopy the beneficiary effect of SMN2 pression Omnibus (GEO) database, www.ncbi.nlm.nih.gov/geo (accession no. GSE83549). gene amplification and compensate for SMN1 deficiency. In ad- 1To whom correspondence should be addressed. Email: [email protected]. SMN1 dition, heterozygotes are asymptomatic, whereas affected This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. homozygotes have 10–20% of normal SMN levels. Therefore, we 1073/pnas.1616521114/-/DCSupplemental.

www.pnas.org/cgi/doi/10.1073/pnas.1616521114 PNAS Early Edition | 1of10 Downloaded by guest on September 25, 2021 of PRC2 activity could lead to increases in SMN2 expression, EZH1 Identification of SMN-Antisense 1 at the SMN Locus. Detailed anal- and EZH2 mRNAs were knocked down in the SMA fibroblast cell ysis of RNA immunoprecipitation-sequencing (RIP-seq) datasets line, GM09677, using antisense oligonucleotides (ASOs) designed revealed a previously undescribed PRC2-interacting antisense for RNaseH-mediated degradation. Two days after transfection of RNA within the mouse Smn locus (17). Here, we investigated both EZH1 and EZH2 gapmers, their respective mRNA levels were whether the antisense transcript exists in humans and may have a significantly decreased by ∼80% each and were associated with an role in PRC2-mediated SMN repression. Next-generation RNA increase in SMN-FL mRNA as shown by reverse transcription sequencing revealed a lncRNA, which we call SMN-Antisense 1 quantitative PCR (RT-qPCR) (Fig.1B). We further analyzed the (SMN-AS1), that is transcribed from the SMN loci (Fig. 2A). Given SMN1 and SMN2 loci (from here on collectively termed “SMN the high sequence identity between the SMN1 and SMN2 loci, we locus”) for chromatin changes upon EZH1/EZH2 knockdown by predict the lncRNA to be transcribed from both loci. As expected, performing ChIP. Because SMN1 and SMN2 have >99% sequence SMN-AS1 was observed in both SMN1- and SMN2-mutated cell lines identity (27,890 of 27,924 match), it is not possible to (Fig. 2B). Furthermore, SMN2 copy number was determined by distinguish between the two chromosomal locations here. We ob- qPCR for carrier and diseased cell lines (18), and we independently served decreased association of EZH2 as well as decreased determined the relative expression of SMN-AS1 and observed a direct H3K27me3 levels at the locus, without any changes in total H3 (Fig. correlation (Fig. 2B). Northern blot analysis of human fetal brain and 1C). This suggests that PRC2 directly regulates the expression of adult lung tissues revealed that SMN-AS1 is up to 10 kb long, is SMN in fibroblasts and potentially other cell types. heterogeneous in size, and has differential expression between the

A Chr. 5 69,340,000 69,345,000 69,350,000 69,355,000 69,360,000 69,365,000 69,370,000 69,375,000 20 _ EZH2 1 _ 20 _

H3K27me3 1 _ HepG2 20 _ input 1 _ SMN2

B EZH1 mRNA EZH2 mRNA 1.2 1.2 2.5 * 1.0 1.0 2.0 0.8 0.8 1.5 lipid 0.6 0.6 1.0 EZH1/EZH2 kd

0.4 0.4 Fold Change Fold Change Fold Change 0.2 0.2 0.5 0 0 0 EZH1 EZH2 SMN-FL lipid lipid EZH1 kd EZH2 kd

C SMN2 10 Kb 12a2b3 4 5 6 7 8 -400 -4200 -1200 +3200 +6700 Exon 7 Exon 6 Exon 5 Exon 8 Exon 1 Exon 2B Exon 2A Exon 3/4

EZH2 H3K27me3 H3 lipid 5 5 8 EZH1/EZH2 kd 4 4 6 3 3 4

% input 2 2 2 1 1 0 0 0 -400 -400 -400 -4200 -1200 -4200 -1200 -1200 -4200 +3200 +6700 +6700 +3200 +3200 +6700 Exon 7 Exon 5 Exon 6 Exon 8 Exon 7 Exon 7 Exon 1 Exon 8 Exon 5 Exon 6 Exon 6 Exon 5 Exon 8 Exon 1 Exon 1 Exon 2B Exon 2A Exon 2B Exon 2B Exon 2A Exon 2A Exon 3/4 Exon 3/4 Exon 3/4

Fig. 1. SMN2 locus is a target of PRC2 regulation. (A) UCSC Genome Browser screen shot of ChIP-sequencing mapped reads for EZH2, H3K27me3, and input at the SMN2 locus from HepG2 cells (reference genome GRCh37/hg19). The plot is a density graph of signal enrichment with a 25-bp overlap at any given site. (B) RT-qPCR for EZH1 and EZH2 mRNA in SMA fibroblast line GM09677 after EZH1 and EZH2 knockdown by transfection of their respective targeting gapmer ASO for 2 d. RT- qPCR for EZH1, EZH2, and SMN-FL mRNA after EZH1 and EZH2 knockdown (mean ± SD; n = 3). *P < 0.05 with one-way ANOVA. (C) Schematic diagram of the SMN2 locus with exons numbered above and with ChIP-qPCR primer positions below (red bars). ChIP-qPCR data for enrichment of EZH2, H3K27me3, and total H3 from EZH1/EZH2 knockdown (green) compared with the lipid transfection control (purple) in the SMA fibroblasts (mean ± ; n = 3).

2of10 | www.pnas.org/cgi/doi/10.1073/pnas.1616521114 Woo et al. Downloaded by guest on September 25, 2021 PNAS PLUS

A 10 Kb B

* carrier disease SMN2 SMN 1/2 1 2a 32b4 5 6 7 8 deletion SMN-AS1 expression 7 7 SMN-AS1 6 SMN2 copy number AS3 6 AS4 5 5 4 4 3 3 2 2 1 1 (Zhong, et al. 2011) SMN2 copy number SMN-AS1 Fold Change 0 0 GM20384 GM10684 GM03814 GM03813 GM009677 GM00232b C D E AS4 AS3 AS3 SMN-FL mRNA SMN-AS1 RNA SMN-AS1 Fold change Fold change SMN mRNA (norm to Adrenal Gland) (norm to Adrenal Gland) SMN pre-mRNA 152025 10 05 01020 30 40 FetalLung brainFetalLung brain WT (brain)5025 (brain) Fetal brain Spinal cord Cerebellum 10 kb SMN-AS1 Whole brain Skeletal muscle Fetal liver Placenta Bone marrow Thymus MEDICAL SCIENCES Uterus 7SK B2M Prostate F Liver Adrenal gland Salivary gland 40 Colon IgG 30 Testes α−SUZ12 Small intestine Spleen 20 Lung % input Kidney 10

0 18S B2M TUG1 RPL19 ANRIL GAPDH SMN-AS1 (set1) SMN-AS1 (set2) G RepA I-IV MBP 1-441 1.0 RepA I-IV 63 250 500 1000 1000 0 250 125 PRC2 [nM] 0 500 63 125 SMN-AS1, PRC2 binding reg bound MBP unbound SMN AS1, non-binding reg SMN-AS1 SMN-AS1 0.5 PRC2 binding reg non-binding reg Fraction bound 0 125 250 500 1000 63 125 250 500 1000 63 0 Rep A I-IV Kd = 358 nM bound SMN-AS1 PRC2 region Kd = 355 unbound 0.0 0 500 1000 PRC2 [nM]

Fig. 2. Identification of a lncRNA at the SMN locus, SMN-AS1.(A) Mapping of SMN-AS1 positioned relative to the SMN genes. The red asterisk marks the location of the C-to-T transition found in SMN2. AS3 and AS4 are Northern blot probes. (B) Correlation of expression and SMN copy number. SMN-AS1 relative expression as measured by RT-qPCR (indicated on the left y axis, blue bars) and SMN2 genomic copy number as measured by digital qPCR (indicated on the right y axis, red bars) in SMA disease fibroblast lines and a carrier line by Zhong et al., 2011 (18). GM20384 cells lacking SMN2, but retaining SMN1,alsoexpressSMN-AS1. (C)NorthernblotofhumanSMN-AS1 from human fetal brain and adult lung tissue detected with AS3 and AS4 probes. In the second Northern blot, WT and 5025 SMA mouse brains were probed with AS3, showing the signal for SMN-AS1 is detected in the 5025 mouse harboring two copies of the human SMN locus. (D)RT- qPCR of SMN-AS1 and SMN-FL mRNA from 20 human tissue types with the fold change normalized to the expression level in the adrenal gland. (E) Strand-specific single-molecule RNA-FISH. Maximum intensity merge of widefield z stack in GM09677 SMA fibroblasts of the nascent SMN pre-mRNA (red, detected by a set of intronic probes), the mature SMN mRNA (yellow), and the SMN-AS1 lncRNA (blue). Pre-mRNA signals are offset (up + left) and mature mRNA signals are offset (down + right) by 2 pixels to enable visualization. (F) Anti-SUZ12 nRIP of SMN-AS1 with two primer sets (set 1 and set 2), TUG1 RNA, ANRIL RNA, 18S rRNA, GAPDH mRNA, beta-2-microglobulin (B2M) mRNA, and RPL19 mRNA from SMA fibroblasts with enrichment shown as % input (mean ± SD; n = 3). IgG nRIP (blue) served as the negative control for the SUZ12 nRIP (red). (G) RNA-EMSA of human PRC2 (EZH2/SUZ12/EED) combined with RepA I–IV, maltose-binding protein (MBP) (1–441), SMN-AS1 (PRC2 binding region), or SMN-AS1 (nonbinding region). Binding curves are displayed at Right (mean ± SD; n = 3).

Woo et al. PNAS Early Edition | 3of10 Downloaded by guest on September 25, 2021 two tissue types (Fig. 2C). To confirm the specificity of the SMN-AS Such gapmer ASOs were used for knockdown of EZH1 and EZH2 probe, we turned to a humanized SMA mouse model carrying two in earlier experiments (Fig. 1C). In contrast to the gapmer ar- copies of the human SMN2 genomic locus (5025 strain) (19). Com- rangement, a “mixmer”-formatted oligo lacks the central DNA paring the brain tissues from wild-type and 5025 mice, we observed a component by the introduction of interspersed chemically modi- similar set of transcripts in the SMN2-harboring transgenic mice fied nucleotides. It does not support the RNaseH-mediated deg- as detected in the human fetal brain (Fig. 2C). By RT-qPCR, we radation but rather functions as a steric blocker (24). We generated detected SMN-AS1 in patient cell lines, and the level of expres- mixmer oligos consisting of LNA interspersed with 2’-O-methyl sion correlated with SMN2 copy number (as determined by ref. 18) nucleotides for high-affinity binding to SMN-AS1. Oligos were (Fig. 2B). In addition, we found that SMN2 mRNA and SMN-AS1 designed to target regions enriched from EZH2-associated RNAs expression is highly correlated, with highest levels in CNS tissues by RIP followed by next-generation RNA sequencing (17). (Fig. 2D). Finally, strand-specific single-molecule RNA-fluorescent in Screening of mixmers led us to focus on one efficacious mixmer, situ hybridization (RNA-FISH) detected expression of SMN-AS1 RN-0005 (Fig. 3A). Transfecting RN-0005 into SMA fibroblasts colocalized with the pre-mRNA transcript at the SMN locus (Fig. 2E) significantly increased SMN-FL expression, whereas transfecting in all SMA fibroblasts (52 out of 52) that were imaged in three in- the control oligo, RN-0012, which hybridizes to a region outside of dependent experiments, suggesting that SMN-AS1 may function in a PRC2 interaction domain, did not change SMN-FL expression cis. We observed primarily one colocalized foci in the SMA fibro- (Fig. 3B). Consistently, nRIP showed that RN-0005, but not the blasts and a wild-type fibroblast line. Although the SMN1 and SMN2 control oligo, disrupted the binding of PRC2 to SMN-AS1,as genes are separated linearly by 900 kb, their physical distance is un- shown by RIP-qPCR (Fig. 3C). Furthermore, oligos targeting known in vivo. Our data suggest that they are close enough in SMN-AS1 did not affect the interactions between PRC2 and proximity in fibroblasts, such that single molecule RNA-FISH would ANRIL, GAPDH,orRPL19 control RNAs. These results were not be able to discern SMN1 from SMN2-derived signals. Together, also observed when nRIP was performed using an antibody against these data demonstrate the presence of an antisense transcript in the EZH2 (SI Appendix,Fig.S2). As expected, single-molecule RNA- SMN1 and SMN2 loci. FISH for the localization of SMN-AS1 after transfection with RN- 0005 showed no change in both the abundance and the localization SMN-AS1 Binds PRC2. To investigate the role of SMN-AS1 in the of SMN-AS1 in ∼90% of cells examined (39 of 42 nuclei) per- PRC2-mediated epigenetic regulation of the SMN2 locus, we formed in three independent experiments (SI Appendix,Fig.S3). performed native RIP (nRIP) using an antibody against the PRC2 Together, these results demonstrate that selective inhibition of subunit SUZ12, followed by RT-qPCR with two distinct probe sets PRC2:SMN-AS1 interaction by a mixmer oligo leads to increased directed to different regions of SMN-AS1. RIP-qPCR showed that SMN2 expression. SMN-AS1 is strongly associated with PRC2 in SMA fibroblasts To gain molecular insight into how the active oligo induced (Fig. 2F). The association was stronger than, or comparable to, SMN expression, we characterized the chromatin changes at the that of well-established PRC2-interacting lncRNAs including SMN locus in response to the disruption of PRC2:SMN-AS1 TUG1 (20) and ANRIL (21). Additionally, PRC2 did not associate interaction using ChIP. When SMA fibroblasts were treated with with highly expressed negative control transcripts such as GAPDH RN-0005, we observed a loss of EZH2 association as well as and RPL19. Similar results were observed with the nRIP for EZH2 decreased H3K27me3 along the SMN gene body (Fig. 3 D and E), (SI Appendix,Fig.S1), further supporting the association of SMN- suggesting that the oligo blocked the recruitment and activity of AS1 with PRC2. Because nRIP identifies both direct and indirect PRC2.Furthermore,therewasan increase in association of RNA interactions, we next performed RNA electrophoretic mobility Pol II-phosphoSer2 (RNA polymerase II, phosphorylated at serine shift assays (RNA EMSAs), which specifically detect direct inter- 2)andelevatedlevelsofH3K36me3, both of which indicate greater actions. Using a 441-nt RNA containing the PRC2-interacting transcriptional elongation (Fig. 3 F and G). By contrast, pan-H3 region of SMN-AS1 (SMN-AS1, PRC2-binding region), as identi- levels were unaffected by treatment (Fig. 3H). H3K4me3, a mark fied by RIP-seq. (17), we observed that purified recombinant hu- of transcription initiation, was enriched at the promoter in the lipid man PRC2 (EED/SUZ12/EZH2) specifically changed its migration control samples. Interestingly, H3K4me3 levels did not change at (Fig. 2G). Binding was concentration-dependent and was as robust the promoter with RN-0005 addition (Fig. 3I), suggesting that the as that of the 434-nt RepA RNA, a conserved domain of XIST increased SMN mRNA levels may be occurring in a setting where RNA that is a well-documented PRC2-interacting lncRNA (17, 22, basal levels of transcription exist. No changes in PRC2 association 23). Dissociation constants (Kd) of both transcripts were estimated were observed at another well-established Polycomb target locus, to be 350–360 nM, suggesting that the association of this lncRNA HOXC13, upon treatment (Fig. 3J). We also performed ChIP on with PRC2 at this site is comparable to the RepA domain of XIST. SMA fibroblasts that were treated with a splice-correcting oligo As specificity controls, we observed a low level of background (SCO), which targets the pre-mRNA for exon 7 inclusion but does binding to a non–PRC2-interacting 441-nt region of the SMN-AS1 not alter the transcription rate at the SMN locus (SI Appendix,Fig. transcript (SMN-AS1, nonbinding region) and to another non- S4). No chromatin changes were observed. These data suggest that specific mRNA of similar length, maltose-binding protein from PRC2 recruitment and histone methyltransferase activity at the Escherichia coli (22). These data lead us to conclude that SMN-AS1 SMN locus can be selectively inhibited by an oligo by mechanisms lncRNA interacts directly and specifically with PRC2. that may include steric blocking of the specific PRC2:SMN-AS1 interaction or disruption of a secondary structure within SMN-AS1 Blocking PRC2:SMN-AS1 Interaction Up-Regulates SMN2 and Produces that would be recognized by PRC2. Epigenetic Changes. To investigate the effect of disrupting PRC2:SMN-AS1 interactions, we designed chemically modi- Blocking PRC2 Recruitment Results in SMN2 Up-Regulation in fied ASOs targeting the PRC2-binding region of the lncRNA Fibroblasts. We further characterized SMN2 mRNA up-regulation, for hybridization via Watson–Crick complementarity pairing. which resulted from the disruption of the PRC2:SMN-AS1 in- Depending on the arrangement of DNA and locked nucleic acid teraction and the subsequent epigenetic changes at the SMN (LNA)-modified nucleotides, such base pairing can lead to either locus. We used the GM09677 fibroblasts, which carry two copies RNaseH-mediated degradation of target RNAs or hindering of of the SMN2 gene and are homozygous for SMN1 exons 7 and 8 the interaction between target RNAs and their binding partners. deletion. RT-qPCR analyses with three different primer sets de- For RNaseH-mediated degradation, a “gapmer”-formatted ASO tected a concentration-dependent increase of various SMN composed of a central DNA segment greater than 6 nucleotides mRNA transcripts, including all SMN isoforms (exon 1–2) as well (i.e., gap) flanked by 2–4 LNA-modified nucleotides is required. as isoforms including or excluding exon 7, SMN-FL,andSMNΔ7,

4of10 | www.pnas.org/cgi/doi/10.1073/pnas.1616521114 Woo et al. Downloaded by guest on September 25, 2021 PNAS PLUS A SMN2 10 Kb 12a2b3 4 5 6 7 8 oligo control RN-0005

B CDp = 0.006 EZH2 45 * 4 lipid 40 3 SMN-FL mRNA mock, IgG RN-0005 35 mock, α-SUZ12 2 8 30 RN-0005, α-SUZ12 % input 1 control oligo, α-SUZ12 6 25 0 20 % Input 4 -400 -1200 -4200 +3200 +6700 Exon 7 Exon 8 Exon 6 Exon 5 15 Exon 1 Exon 2B Exon 2A 2 Exon 3/4

Fold change 10 0 5 0

RN-0005 ANRIL RPL19 control oligo GAPDH SMN-AS1 SMN mRNA RNA Pol II phospho-Ser2 H3K36me3 H3K27me3 8 EF5 G12 lipid 10 MEDICAL SCIENCES 4 6 RN-0005 8 3 4 6 2

% input 4 1 2 2 0 0 0 -400 -400 -4200 -1200 -400 +6700 +3200 -4200 -1200 Exon 7 Exon 8 Exon 5 Exon 6 Exon 1 +3200 +6700 -1200 -4200 +3200 +6700 Exon 7 Exon 8 Exon 6 Exon 5 Exon 1 Exon 7 Exon 6 Exon 8 Exon 5 Exon 1 Exon 2B Exon 2A Exon 3/4 Exon 2B Exon 2A Exon 3/4 Exon 2B Exon 2A Exon 3/4

H3 H3K4me3 H 8 I 12 J HOXC13 promoter 9 10 lipid 6 8 8 7 RN-0005 4 6 6 5 % input 4 2 4 2 3 % input 2 0 0 1 0 -400 -400 -4200 -1200 -4200 -1200 +6700 +3200 +3200 +6700 Exon 7 Exon 7 Exon 5 Exon 6 Exon 8 Exon 5 Exon 6 Exon 8 Exon 1 Exon 1 H3 EZH2 Exon 2B Exon 2B Exon 2A Exon 2A Exon 3/4 Exon 3/4

H3K27me3A PolII pS2H3K36me3 H3K4me3 RN

Fig. 3. PRC2 is associated with SMN-AS1 and selective dissociation leads to PRC2 loss and chromatin changes at the SMN locus. (A) Schematic diagram of the SMN2 locus with exons numbered above and mixmer oligo positions (squares) below. (B) RT-qPCR of SMN-FL mRNA after transfection with RN-0005 or RN-0012 in SMA fibroblasts for 3 d. (C) Anti-SUZ12 nRIP of SMN-AS1, ANRIL, GAPDH mRNA, and 18S rRNA from SMA fibroblasts after lipid (red) or RN-0005 (green) or RN-0012 (blue) transfection; IgG RIP (purple) (mean ± SD; n = 3). *P < 0.05 using two-tailed Student’s t test. (D–I) ChIP at the SMN2 locus in GM09677 SMA fibroblasts that were transfected with lipid (purple) or RN-0005 (green) using antibodies against (D) EZH2, (E) H3K27me3, (F) RNA polymerase II phospho-Serine2, (G) H3K36me3, (H) pan-H3, and (I) H3K4me3 (mean ± SD; n = 3). (J) ChIP at the promoter of HOXC13, a PRC2-regulated gene, for EZH2, H3K27me3, RNA polymerase II, phospho-Serine 2 (RNA PolIIpS2), H3K36me3, H3, and H3K4me3, after transfection with lipid or RN-0005 in SMA fibroblasts. (mean ± SD; n = 3).

respectively (Fig. 4A). Furthermore, overall SMN protein levels of PRC2 with its recruiting lncRNA resulted in up-regulation of also increased, as shown by ELISA after 5 d of treatment, which both SMN mRNA and protein. supports the epigenetic evidence of increased transcription (Fig. To determine how targeting the disruption of PRC2:SMN-AS1 4B). Western blot results revealed that this increase could be at- interactions might affect PRC2 targets globally, we performed tributed to the 38-kDa SMN protein (Fig. 4C). Both quantification RNA sequencing from samples with specific disruption of of ELISA and Western analyses indicated an approximately PRC2:SMN-AS1 (using RN-0005) or global inactivation of the fourfold protein up-regulation following treatment in SMA fi- PRC2 complex (using a SUZ12 gapmer). Treatment with either broblasts with RN-0005. Taken together, blocking the interaction RN-0005 or the SUZ12 gapmer resulted in significant increases

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12a2b3 4 5 6 7 8 SMN2

exons 1-2 FL

SMN exons 1-2 mRNA 7SMNmRNA 8 4 10 SMN-FL mRNA

8 6 3 6 4 2 4 Fold Change Fold Change 2 Fold Change 1 2

0 0 0 0.1 1 10 100 1000 0.1 1 10 100 1000 0.1 1 10 100 1000 Oligo Concentration (nM) Oligo Concentration (nM) Oligo Concentration (nM)

BCSMN protein - ELISA SMN protein - western blot 8 Conc (nM) 5

6 4 6.25 50 lipid 25 MWM cells 12.5 αtubulin (55 kD) 3 4 2 2 SMN (38 kD)

Fold Change EC50 = 5 nM 1 Fold change 0 0 0.1 1 10 100 1000 110100 Oligo Concentration (nM) Oligo Concentration (nM)

D SMN-FL mRNA 20 3 SMN Pathway analysis ** Bin Size * 10 2 30 RN-0005 20 1 0 14 138 10 21 Fold Change

0

RN-0005 vs mock (t-statistic) -10 Mock SUZ12 gapmer SUZ12 -20 -10 0 10 RN-0005 gapmer SUZ12 kd gapmer vs mock (t-statistic)

Fig. 4. Up-regulation of SMN expression upon RN-0005 treatment. (A) RT-qPCR of SMN (exon 1–2), SMN Δ7, and SMN-FL mRNA in GM09677 SMA fibroblasts (mean ± SD; n = 5). (B) Changes in total SMN protein levels after SMA fibroblasts were transfected with RN-0005 for 5 d (mean ± SD; n = 3), measured by ELISA. (C) Western blot results for SMN and α-tubulin in SMA fibroblasts after SMA fibroblasts were transfected with RN-0005 for 5 d (mean ± SD; n = 2). (D) RT-qPCR of SMN-FL mRNA in GM09677 fibroblasts that were transfected with 15 nM RN-0005 or 15 nM SUZ12 gapmer ASO (mean ± SD; n = 4). *P < 0.05, **P < 0.01 using one-way ANOVA. A hexagonally binned scatterplot of the moderated t statistics of the 11,887 annotated genes tested for differential expression posttreatment with RN-0005 or the SUZ12 knockdown ASO. Each bin is colored by the number of genes that fall within it, showing the trend of RN- 0005–treated t statistics (and those less significantly differentially expressed genes) generally being reduced compared with their SUZ12 knockdown ASO counterpart t statistics. The Venn diagram shows the significant results (q < 0.10) of the pathway analysis using competitive gene set tests on 1,281 canonical pathways after treatment with each oligo. Overlap required that a pathway was significantly affected in the same direction by both oligos. There is significant overlap between the oligo treatments when tested with a hypergeometric test (P = 1.36e−11), however ∼4.5 times more pathway gene sets were significantly changing with SUZ12 KD treatment.

in SMN mRNA levels compared with transfection control samples SUZ12 knockdown (KD) t statistic is usually larger than their (Fig. 4D). Globally, there were approximately fourfold more gene respective RN-0005 t statistic. As this is a scatterplot encom- expression changes with the SUZ12 gapmer treatment than with passing all genes and we expect that most genes do not change RN-0005 treatment that had at least a 1.5-fold change (q < 0.05). significantly or have a large magnitude of change, the two gene This is depicted by a scatterplot of the moderated t statistics of the expression profiles correlate overall. Here, a strong linear cor- gene expression changes where for most genes, their individual relation was observed while simultaneously many more significant

6of10 | www.pnas.org/cgi/doi/10.1073/pnas.1616521114 Woo et al. Downloaded by guest on September 25, 2021 changes occurred with the SUZ12 gapmer treatment. Looking treatment, suggesting fewer changes overall. Taken together, this PNAS PLUS closer at expression profiles of genes neighboring SMN1 and suggests that RN-0005 has a more localized effect than the more SMN2, the nearest neighboring genes that changed significantly global effects of knocking down PRC2. Pathway gene set analyses in response to RN-0005 treatment were ADAMTS6 (upstream) identified significant pathways (10% false discovery rate) with each and BDP1(downstream) at 4.6 Mb and 1.4 Mb away from SMN2, oligo treatment, and although there were overlaps between the respectively. As the closest changed genes are greater than a datasets, many more pathways changed separately with SUZ12 megabase away, these data suggest PRC2:SMN-AS1 regulation is knockdown (Fig. 4D and SI Appendix,Fig.S5). This suggests that localized to the SMN locus. In contrast, the nearest significant although there is a relationship between the downstream genes neighbor genes that changed after SUZ12 knockdown were effected by modulating SMN expression, there are less overall TAF9, 0.8 Mb upstream, and BDP1, 1.4 Mb downstream, of downstream changes when you up-regulate SMN expression with SMN2. Furthermore, because both RN-0005 and the SUZ12 KD RN-0005 than when you target SMN through a global knockdown gapmer do affect SMN expression, we would expect to see po- of PRC2. tential overlap in downstream changes, which we confirm by identifying 21 pathways overlapping between oligo treatments Blocking PRC2 Recruitment Results in SMN2 Up-Regulation in Neuronal (only approximately 4–5 overlaps would be expected by chance if Cultures. Although SMN expression is ubiquitous, its expression is there was no relationship between the two treatments). However, highest in the central nervous system (Fig. 2D) (25), particularly in we see far fewer pathways modulated significantly with RN-0005 spinal motor neurons where the disease is manifested (5, 6, 26).

SMA iPS-derived motor neuron cultures SMA iPS-derived motor neuron cultures SMA iPS-derived motor neuron cultures A SMN-FL mRNA SMN-FL mRNA (day 7) SMN-FL mRNA 3 2.0 2.0 ** untreated * RN-0005 1.5 1.5 2 inactive oligo unrelated oligo 1.0 1.0 RN-0005 1 Fold change Fold change Fold change 0.5 0.5 MEDICAL SCIENCES 0 0 0 10 uM 317 9 1 uM uM 5 20 untreated

BCmouse cortical neurons D mouse cortical neurons human SMN-FL mRNA mock RN-0027 (10 μM) human SMN-FL mRNA 4 * untreated 5 Day 7 RN-0027 3 * 4 Day 14 control oligo day 14 3 Day 18 2 2 1 Fold change Fold change 1 untreated 0 0 0.1 1 10 10 0 1 uM 3 uM 10 uM untreated EZH2 gapmer (μM)

E human SMN-FL mRNA F human SMN protein 6 SC + mixmer 6 SC + mixmer SC alone SC alone 4 4

2 2 Fold change mixmer alone mixmer alone Fold change (SMN/GAPDH)

0 0 Mock 0.01 0.1 1 10 0.01 0.1 1 10 Splice corrector (μM) Splice corrector (μM)

Fig. 5. Distinct mechanisms of SMN-FL mRNA generation can be complementary in neuronal cultures. (A, Left) RT-qPCR of SMN-FL mRNA in human SMA iPS- derived motor neuron cultures after gymnotic treatment with RN-0005, an inactive oligo, or an unrelated oligo at 5 and 20 μM for 11 d as a fold change from untreated cells (mean ± SD; n = 3). **P < 0.01 using one-way ANOVA. (Middle) RT-qPCR of SMN-FL mRNA in human SMA iPS-derived motor neuron cultures at day 7 after treatment with an EZH2 gapmer ASO (mean ± SD; n = 2). *P < 0.05 using one-way ANOVA. (Right) RT-qPCR for SMN-FL mRNA in human SMA iPS- derived motor neuron cultures after gymnotic treatment with RN-0005 at 20 μM for 3, 7, 9, or 11 d as a fold change from untreated cells (mean ± SD; n = 2). (B) Images of 5025 mouse cortical neurons at day 14 either mock-treated or treated with RN-0027 at 10 μM. (Scale bar, 100 μm.) (C) RT-qPCR of human SMN-FL mRNA relative to mouse gusb mRNA from the 5025 mouse cortical neurons that were treated with either 1.1, 3.3, or 10 μM RN-0027 (mean ± SD; n = 5) or control oligo (mean ± SD; n = 2). *P < 0.05 using two-way ANOVA. (D) RT-qPCR of human SMN-FL mRNA relative to mouse gusb mRNA from the 5025 mouse cortical neurons that were treated with 0.1, 0.3, 1.1, 3.3, or 10 μM EZH2 gapmer for 14 d (mean ± SD; n = 2). (E) RT-qPCR results of human SMN-FL mRNA from SMA mouse from 5025WT SMA mouse model cortical neurons that were treated with a fixed concentration of RN-0027 (10 μM) in combination with in- creasing concentrations of a SCO for 14 d (mean ± SD; n = 2). (F) Human SMN protein levels from 5025 SMA mouse cortical neurons that were treated with a fixed RN-0027 concentration in combination with increasing concentrations of a SCO for 14 d (mean ± SD; n = 2), as measured by ELISA.

Woo et al. PNAS Early Edition | 7of10 Downloaded by guest on September 25, 2021 To assess the activity of RN-0005 in disease-relevant cells, we Discussion examined SMN expression in two neuronal cell types. First, we Treatments for SMA are focused on addressing symptoms generated induced pluripotent stem cells (iPSCs) derived from + ranging from respiratory complications to muscle atrophy. Various SMA patient fibroblasts and differentiated them into SMI32 approaches to treat SMA are being tested in clinical trials to ad- motor neurons (SI Appendix,Fig.S6). After treating with RN-0005 dress both neurological and muscular decline (reviewed by ref. 32). SMN-FL for 14 d, mRNA displayed a statistically significant Splice-correcting therapies use ASOs or small molecules to pro- twofold increase relative to untreated or control-treated motor mote exon 7 inclusion. Recently, an ASO which includes exon 7 by A SMN-AS1 neurons (Fig. 5 ). Both an inactive oligo that targets the splice-correcting approach, Spinraza, was approved by the SMN but does not up-regulate mRNA and an unrelated oligo that FDA. Gene therapy replacement of SMN1 offers an alternative SMN-AS1 SMN does not target showed no effect on mRNA levels. strategy to increase levels of SMN protein and is currently being As expected, EZH2 knockdown also led to a similar increase in tested. A neuroprotective agent may offer some resilience to motor SMN-FL mRNA. We performed a time-course experiment with neurons, but these agents have not proven effective in other neu- SMN-FL RN-0005 and observed a delayed increase in mRNA rodegenerative disorders such as ALS. A skeletal muscle enhancer levels in neurons relative to what was seen in fibroblasts. This may is being evaluated to determine whether protecting the muscle will be partially due to the mode of delivery (unassisted delivery versus lessen disease progression. transfection) and/or the nonproliferating state of the neuronal cells We report a transcriptional up-regulation method to selec- versus the highly proliferative fibroblasts. Supporting the latter, tively up-regulate endogenous SMN mRNA and protein with the the rate of H3K27me3 removal from chromatin of nondividing identification and characterization of an lncRNA associated with cells is slower than in proliferating cells (27). Taken together, the SMN1 and SMN2 loci. The two genes are nearly identical in these data show that disrupting the PRC2:SMN-AS1 interaction SMN sequence, resulting from a chromosomal duplication, which would leads to up-regulation in disease-relevant and postmitotic suggest that their regulation might be the same. We showed that in motor neuronal cultures. both SMN1-andSMN2-mutated cell lines, SMN-AS1 is expressed. We also prepared primary cortical neuronal cells from E14 Moreover, SMN2 copy number in patient cell lines correlated with embryos of the 5025 SMA mice and treated them with a chemical SMN-AS1 SMN-AS1 the relative expression level of , suggesting that this variant of RN-0005 that targets the same sequence and lncRNA is tightly associated with each copy of the gene. Taken shows similar blocking of PRC2:SMN-AS1 and mRNA up-regu- together, it is likely that SMN-AS1 regulation of expression of both lation (SI Appendix,Fig.S7) but has a more favorable in vivo safety genes might be similar. It remains possible that other mecha- profile. RN-0027 was added to the neurons for 14 d without ob- nisms may contribute to regulating SMN1 and SMN2 expression vious toxicity or changes in cell morphology (Fig. 5B). We observed differentially, perhaps temporally or spatially, but we are unable a concentration-dependent increase in SMN-FL mRNA with a to distinguish between them with our sequenced-based assays. threefold increase at 10 μM following 14 d of treatment (Fig. 5C) Therefore, we address the two loci as one. in multiple experiments. Furthermore, the addition of a control Disruption of the lncRNA:PRC2 interaction resulted in oligo did not result in changes in SMN-FL levels. In agreement changes to SMN expression but not to expression changes in the with the results obtained from patient fibroblasts (Fig. 1B)and neighboring genes based on our RNA-seq data, suggesting that motor neuron cultures (Fig. 5A), ex vivo cortical neurons treated with an EZH2 gapmer ASO displayed a concentration-dependent the lncRNA functions in cis. The overall changes in PRC2 and SMN-FL D RNA polymerase II occupancy and histone modifications suggest increase in mRNA levels (Fig. 5 ). Our findings from ex SMN2 vivo cortical neurons suggest that there is in vivo relevance of this that the increase in steady-state levels of arises at the mechanism in terminally differentiated neuronal cells. transcriptional level in disease-relevant cell types. Indeed, when mouse primary cortical neurons carrying copies of human SMN2 Combination of Transcriptional Up-Regulation and SMN Exon 7 Splice were treated with our transcription-activating mixmers and a SMN2 Correction Increases SMN-FL mRNA. Splice-correcting modifiers SCO, we observed an additive effect of increased ex- have been designed to facilitate the inclusion of exon 7 during pression beyond that offered by a splice-correcting therapy alone SMN2 transcription (28), resulting in the production of SMN-FL to potentially confer greater therapeutic benefit. mRNA and functional SMN protein. Although steady-state total LncRNAs isolated by nuclear fractionation were shown to be SMN mRNA levels would not increase with a splice-correcting tethered to neighboring protein-coding genes (33). LncRNAs modifier, the shift to increase SMN-FL mRNA levels has been have diverse cellular functions and are critical for maintaining demonstrated to be beneficial to survival in mice (29, 30) and in cellular identity (34). Furthermore, it has been demonstrated humans (31). Because the transcriptional activation approach up- that PRC2 is associated with lncRNAs, and it has been suggested regulates SMN through a distinct mechanism from that of a splice that this relationship may serve to recruit PRC2 to specific sites corrector, we reason that combining these two mechanisms will be (17, 35). In vitro studies and a recent study using iCLIP for PRC2 more effective than with a single approach. To this end, the 5025 have demonstrated that PRC2 interacts with RNAs nonspecifically mouse cortical neurons, which only harbored human SMN2 and and that nascent transcripts may divert PRC2 from being recruited not human SMN1, were treated with either a SCO, our transcrip- to an actively transcribed gene (36). However, this does not ex- tional activating mixmer, or a combination of the two oligos for clude the possibility that lncRNAs may also interact with PRC2 14 d to measure the levels of SMN-FL mRNA (Fig. 5E). Although through specific interactions. Our data demonstrate transcriptional treatment with the SCO alone resulted in a 2–3-fold increase in up-regulation of SMN resulting from the loss of PRC2 association SMN-FL mRNA, an additional 1.8-fold increase was observed in with the chromatin by targeting an oligo to disrupt a specific combination with the mixmer treatment. This additive effect was lncRNA: PRC2 interaction site. We believe that this interaction is also detected with an increase in the human SMN protein levels by specific as well, as the disruption of SMN-AS1 with PRC2 does not a human-specific ELISA (Fig. 5F). Although the SCO up-regulated change the association of other lncRNAs with PRC2, and we see SMN protein levels ∼2.5-fold, the combination resulted in a fewer pathway and expression changes compared with directly fourfold increase of SMN levels. These data further provide evi- knocking down PRC2 to increase SMN expression. dence that SMN-AS1 inhibition increases SMN-FL mRNA and In summary, we have demonstrated proof-of-concept that our SMN protein levels by a mechanism that is independent and gene up-regulation technology disrupts the interaction between complementary to that of splice correction. The increased SMN-FL PRC2 and a lncRNA, which leads to the increased expression of mRNA and protein resulting from the combination approach may its associated protein-coding gene. Our approach of preventing provide greater benefit in treating SMA. PRC2 recruitment to a specific genomic location potentially

8of10 | www.pnas.org/cgi/doi/10.1073/pnas.1616521114 Woo et al. Downloaded by guest on September 25, 2021 offers greater selectivity and elicits fewer unintended side effects transfected at 30 nM with Lipofectamine 2000 at a final volume of 20 mL. Cells PNAS PLUS than small-molecule EZH1/2 inhibitors. Notably, this technology were harvested 3 d posttransfection. achieves the degree of SMN up-regulation considered to be therapeutic for SMA. With this proof-of-concept, we believe that RIP. RIP was performed using the Magna RIP RNA-Binding Protein Immu- our up-regulation platform could be applied to many other dis- noprecipitation Kit (EMD Millipore) using ChIP-grade anti-SUZ12 (Abcam), anti-EZH2 (Abcam), and anti-SETD2 (USBiological Life Sciences) antibodies. eases in which a desirable gene is epigenetically silenced by a RNA was extracted with TRIzol (Life Technologies) and transcribed to cDNA transcriptional repressive complex. using the High Capacity cDNA Reverse Transcription Kit (Applied Biosystems). qPCR was performed on a StepOnePlus Real Time PCR System (Applied Experimental Procedures Biosystems) using TaqMan Fast Advanced Master mix (Applied Biosystems). RNA Sequencing. RNA from GM09677 fibroblasts that were transfected with RN-0005, SUZ12 gapmer ASO, and lipid controls were sequenced (300 bp EMSA. DNA templates for EMSA probes containing T7 promoter sequences paired-end) on the NextSeq500 using Illumina TruSeq stranded total RNA-seq were generated by PCR using Phusion High Fidelity DNA Polymerase (NEB). library preparation kits. Refer to SI Appendix, SI Experimental Procedures for The specific primer sequences are listed in SI Appendix, Table S1.EMSAswere a detailed explanation of the differential gene expression analyses. performed as described previously (Cifuentes-Rojas et al., 2014) (22). RNA probes were transcribed using the AmpliScribe T7 Flash Transcription Kit Northern Blots. (Epicentre) and PAGE purified from 6% (vol/vol) TBE urea gel. RNA probes RNA preparation. Total RNA from human fetal brain and lung tissue was were then dephosphorylated by calf intestinal alkaline phosphatase (NEB), obtained from Clontech and treated with RiboMinus (Life Technologies). We purified by phenol-chloroform extraction, 5′ end-labeled with T4 Polynucleotide fractionated 500 ng of rRNA-depleted RNA on a 1% agarose gel in 1× Mops Kinase (NEB) and [γ-32P] ATP (Perkin-Elmer), and purified with Illustra MicroSpin buffer. RNA was capillary-transferred to BrightStar Plus nylon membrane G-50 columns (GE Life Sciences). RNA probes were folded in 10 mM Tris pH 8.0, (Ambion) overnight in 20× SSC buffer, then cross-linked by UV exposure. For 1 mM EDTA, and 300 mM NaCl by heating to 95 °C, followed by incubations

mouse Northern blots, RNA was isolated from 5025 WT brain tissue and WT at 37 °C and at room temperature for 10 min each. MgCl2 and Hepes pH 7.5 brain tissue and treated with RiboMinus as above. Approximately 750 ng were then added to 10 mM each, and probes were put on ice. We mixed 1 μL RNA was loaded per lane. of 2,000 cpm/mL (2 nM final concentration) folded RNA with PRC2 (EZH2/ Probe preparation. DNA templates containing a T7 promoter for in vitro SUZ12/EED; BPS Bioscience) at the indicated concentration and 50 ng/mL yeast synthesis of radiolabeled RNA probes were generated by PCR from a human tRNA (Ambion) in 20 μL final concentration of binding buffer [50 mM Tris·HCl fetal brain cDNA library or mouse brain cDNA library with primer pairs listed pH 8.0, 100 mM NaCl, 5 mM MgCl2, 10 mg/mL BSA, 0.05% Nonidet P-40, 1 mM in SI Appendix, Table S1. DTT, 20 U RNaseOUT (Invitrogen), and 5% (vol/vol) glycerol]. Binding reactions were incubated for 20 min at 30 °C and applied on a 0.4% hyperstrength Stellaris RNA-FISH. Probe sets were designed against genomic regions listed in agarose (Sigma) gel in THEM buffer (66 mM Hepes, 34 mM Tris, 0.1 mM MEDICAL SCIENCES SI Appendix, Table S1. They were labeled with Quasar 570 (SMN1/2 exons), disodium EDTA, and 10 mM MgCl2). Gels were run for 1 h at 130 V with buffer Quasar 670 (SMN1/2 introns), and Cal Fluor Red 610 (SMN1/2-AS1). Stellaris recirculation at 4 °C, dried, and exposed to a phosphorimager screen. Screens RNA-FISH was performed as described in the Alternative Protocol for Ad- were scanned in a Storm 860 phosphorimager (Molecular Dynamics), and data herent Cells (UI-207267 Rev. 1.0) with the following modifications: 12-mm were quantified by Quantity One and normalized as described (37). KDswere diameter coverslips were used. We used 25 μL hybridization solution with a calculated with Graphpad Prism by fitting the data to a one site-specific final concentration of each probe set of 250 nM. The wash buffer volumes binding model. were halved. The FITC, Cy3, Cy3.5, and Cy5.5 channels were used to capture the signals from each probe set, and the FITC channel was used to identify Western Blot. Cells were lysed 5 d posttransfection using the extraction buffer cellular autofluorescence. The filter sets from Chroma were 49001-ET-FITC, from the SMN ELISA kit (Enzo) with Protease inhibitor mixture tablets SP102v1-Cy3, SP103v2-Cy3.5, and 41023-Cy5.5. The exposure times were 1 s for (Roche). Total protein content was determined with the total BCA assay FITC, Quasar 570, and Cal Fluor Red 610 and 2 s for Quasar 670. GM09677 (Promega) for equal loading. Samples and Hi Mark prestained ladder (Invi- Human Eye Lens Fibroblast (Coriell) adherent cells were grown in Eagle’s trogen) were run on a 4% (vol/vol) Bis–Tris gel, and were transferred Minimum Essential Medium (EMEM) (ATCC) in a humidified 37 °C incubator at to nitrocellulose membrane. The membrane was incubated in blocking

5% CO2 in ambient air. F-12K and EMEM media were supplemented with buffer (Licor) overnight at 4 °C. The SMN antibody (BD catalog no. 610646), 10% (vol/vol) FBS (Fisher Product number SH30071.03) and 5 mL of Pen/ Alpha tubulin antibody (Abcam catalog no. ab125267), and secondary anti- Strep (Life Technologies). F-12 was further supplemented with Normocin mouse and anti-rabbit 800 (Licor) were used, and the membrane was scanned (InvivoGen). Cells were grown on 12-mm microscope circular cover glass with Odessey (Licor). Band intensities for SMN-FL protein and α-tubulin were No. 1 (Fisher #12–545-80) in 24-well flat-bottom cell culture plates (E&K). quantified using Image Studio software. SMA fibroblasts were transfected at 70% confluence by using oligonucleo- tides complexed with Invitrogen Lipofectamine 3000 (Pub Part #100022234, ELISA. GM09677 fibroblasts were plated on a 24-well tissue culture plate at 4 × Pub #MAN0009872, Rev. B.0) and fixed after 2 d. We used 2 ng DNA and 4 μL 104 cells per well in MEM containing 10% (vol/vol) FBS and 1× nonessential P3000 reagent per 50 μL of DNA master mix. We used 0.375 μL Lipofectamine amino acids. Fibroblasts were treated with oligonucleotides the following day. 3000 reagent per 25 μLofOpti-MEM. After 5 d, cells were lysed and protein was quantified with the SMN ELISA Kit (Enzo Life Sciences, Inc.) and normalized to total protein content as deter- RT-qPCR. Total RNA from 20 human tissues (Clontech) were used for cDNA mined by Micro BCA Protein Assay Kit (Thermo Scientific). For the human- synthesis using High Capacity cDNA Reverse Transcription Kit (Applied Bio- specific ELISA used with the cortical neurons, a similar protocol was used. systems). Data of RT-qPCR SMN-AS1 levels were normalized to levels from the Briefly, cells were washed in cold PBS and lysed in RIPA buffer supplemented adrenal gland. GM09677 fibroblasts were plated on a 24-well tissue culture with protease inhibitor Complete Tablets, mini EDTA-free EASYpack (Roche). plate at 4 × 104 cells per well in MEM containing 10% (vol/vol) FBS and 1× Lysates were quantified by BCA, and ∼20–30 μg were used. A mouse mono- nonessential amino acids. Fibroblasts were treated with oligos the following clonal anti-SMN antibody was captured on high binding plates (Pierce) at 1 μg/mL; day. After 2 d, cells were lysed and mRNA was purified using E-Z 96 Total RNA after blocking with BSA in PBS-0.05% Tween-20, lysates were incubated for Kit (Omega Bio-Tek). SMA iPS-derived motor neurons were lysed with 2 h at room temperature; a rabbit polyclonal human SMN-specific anti- TRIzol for RNA isolation per the manufacturer’s protocol. RNA from mouse body at 1 μg/mL was used for detection, followed by HRP-goat anti-rabbit cortical neurons was extracted using the RNeasy kit (Qiagen) per the (Invitrogen). Signal was measured with SuperSignal ELISA PICO chemilu- manufacturer’s protocol. All cDNAs were synthesized using High Capacity minescent substrate (Thermo). Total GAPDH in the lysates was also quan- cDNA Reverse Transcription Kit (Applied Biosystems). SMN-FL, SMN Δ7, tified by ELISA (R&D Systems); SMN protein concentration was normalized SMN Exon 1–2andGUSB mRNA expression was quantified by predesigned to total GAPDH content. TaqMan real-time PCR assays. A list of custom-designed real-time PCR assays is listed in SI Appendix, Table S1. Cortical Neuron Isolation. Brains were isolated from E14 SMNΔ7 embryos and the cortex was dissected with the MACS neuronal tissue dissociation kit Oligonucleotide Transfection. SMA fibroblasts were transfected at 70% con- (Miltenyi Biotec). The collected cortical neurons were plated at 0.5 × 106 cells fluence by using oligonucleotides complexed with Lipofectamine 2000 (Invi- per well in Neurobasal media (ThermoFisher), B-27 supplement (Thermo- trogen) following the protocol suggested by the manufacturer in the 96-well Fisher), and GlutaMax (ThermoFisher) in a 24-well plate coated with poly–D-

and 24-well format. For ChIP, cells were transfected in 15-cm plates and were lysine (Fisher). Cells were incubated at 37 °C, 5% CO2 for 4 d, allowing the

Woo et al. PNAS Early Edition | 9of10 Downloaded by guest on September 25, 2021 cells to mature and networks to form before unassisted delivery of RN-0005. BDNF (10 ng/mL), and GDNF (10 ng/mL). RN-0005 treatments were carried out After 14 d, the cells were harvested for RNA isolation. during this terminal differentiation period. Antibodies used for immunocy- tochemistry were as follows: SSEA4 and SOX2 (Millipore); TRA-1–60, TRA-1–81, β iPS Cell Culturing and Motor Neuron Differentiation. SMA patient and control OCT4, and NANOG (Stemgent); TuJ1 ( 3-tubulin) and Map2 a/b (Sigma); ISLET1 subject dermal fibroblasts or lymphoblastoid cell lines (LCLs) were obtained (R&D Systems); and SMI32 (Covance). from the Coriell Institute for Medical Research. All of the cell lines and protocols in the present study were carried out in accordance with the guidelines ap- ChIP. Cells were cross-linked with 1% formaldehyde for 10 min at room proved by the Stem Cell Research Oversight Committee (SCRO) and Institutional temperature and then quenched with glycine. Chromatin was prepared and Review Board (IRB) at the Cedars–Sinai Medical Center under the auspice IRB- sonicated (Covaris S200) to a size range of 300–500 bp. Antibodies for H3, SCRO Protocols Pro00032834 (iPSC Core Repository and Stem Cell Program) and H3K27me3, H3K36me3, EZH2, and RNA polymerase II Serine 2 (Abcam) and Pro00024839 and Pro00036896 (Sareen Stem Cell Program). The iPSCs were H3K4me3 (Millipore) were coupled to Protein G magnetic beads (NEB), grown to near confluence under normal maintenance conditions before the washed, and then resuspended in IP blocking buffer. Chromatin lysates were start of the differentiation as per protocols described previously (38). Briefly, added to the beads and immunoprecipitated overnight at 4 °C. Antibodies IPSCs were then gently lifted by Accutase treatment for 5 min at 37 °C. We against H3, H3K36me3, RNA polymerase II phosphoserine 2, H3K27me3, and subsequently placed 1.5–2.5 × 104 cells in each well of a 384-well plate in de- EZH2 were obtained from Abcam, and H3K4m3 antibody was obtained from fined neural differentiation medium with dual-SMAD inhibition (39). After 2 d, Millipore. We used 10 μg of antibody per IP. IPs were washed, RNase A (Roche) neural aggregates were transferred to low adherence flasks. Subsequently, treated, and Proteinase K treated (Roche), and the cross-links were reversed by neural aggregates were plated onto laminin-coated six-well plates to induce incubation overnight at 65 °C. DNA was purified, precipitated, and resus- rosette formation in media supplemented with 0.1 μM retinoic acid and 1 μM pended in nuclease-free water. Custom TaqMan probe sets were used to de- puromorphine along with 20 ng/mL BDNF, 200 ng/mL ascorbic acid, 20 ng/mL termine enrichment of DNA. Probes were designed using the custom design GDNF, and 1 mM dbcAMP. Neural rosettes were isolated, and the purified tool on the Life Technologies website. Primer sequences are listed in SI Ap- rosettes were subsequently supplemented with 100 ng/mL of EGF and FGF. pendix, Table S1. These neural aggregates, termed iPSC-derived motor neuron precursor All RNA-sequencing data were deposited in the Gene Expression Omnibus spheres (iMPSs), were expanded over a 5-wk period. For terminal differ- (GEO) database under accession no. GSE83549. entiation, iMPSs were disassociated with Accutase and then plated onto laminin-coated plates over a 21-d period before harvest using the MN ACKNOWLEDGMENTS. We thank Jeannie T. Lee, Andrey Sivachenko, and maturation media consisting of Neurobasal supplemented with 1% N2, Jonathan C. Cherry for critical discussions and Jonathan Cherry and Brian ascorbic acid (200 ng/mL), dibutyryl cyclic adenosine monophosphate (1 μM), Bettencourt for reading of the manuscript.

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