SUPPLEMENTARY INFORMATION SMN2 Splice Modulators Enhance ... · SUPPLEMENTARY INFORMATION SMN2...

SUPPLEMENTARY INFORMATION

SMN2 Splice Modulators Enhance U1/pre-mRNA Association and Rescue SMA Mice

James Palacino1,3, Susanne E. Swalley1,3*, Cheng Song1,3, Atwood K. Cheung1, Lei Shu1, Xiaolu

Zhang1, Mailin Van Hoosear1, Youngah Shin1, Donovan N. Chin1, Caroline Gubser Keller2,

Martin Beibel2, Nicole A. Renaud1, Thomas M. Smith1, Michael Salcius1, Xiaoying Shi1, Marc

Hild1, Rebecca Servais1, Monish Jain1, Lin Deng1, Caroline Bullock1, Michael McLellan1, Sven

Schuierer2, Leo Murphy1, Marcel J.J. Blommers2, Cecile Blaustein1, Frada Berenshteyn1,

Arnaud Lacoste1, Jason R. Thomas1, Guglielmo Roma2, Gregory A. Michaud1, Brian S. Tseng1,

Jeffery A. Porter1, Vic E. Myer1, John A. Tallarico1, Lawrence G. Hamann1, Daniel Curtis1,

Mark C. Fishman1, William F. Dietrich1, Natalie A. Dales1 & Rajeev Sivasankaran1*

1Novartis Institutes for Biomedical Research, Cambridge, MA, USA and 2Basel, Switzerland.

3These authors contributed equally to this work

*Correspondence and requests for materials should be addressed to S.E.S.

([email protected]) and R.S. ([email protected]).

Nature Chemical Biology: doi:10.1038/nchembio.1837

‐ S2 -

SUPPLEMENTARY RESULTS

Supplementary Table 1 Small molecule screening data

Supplementary Table 2 Candidate genes and exons identified in RNAseq

Supplementary Figure 1 In vitro activity and in vivo pharmacokinetics

Supplementary Figure 2 NVS-SM1 activity in additional cellular models

Supplementary Figure 3 Body weight analysis of NVS-SM1 dosing holiday in SMN7 mice

Supplementary Figure 4 Exemplar RNAseq exon transcript and junction variants

Supplementary Figure 5 High-throughput qPCR validation of RNAseq splicing candidates

Supplementary Figure 6 SMN2-BRCA minigene splicing data with NVS-SM2

Supplementary Figure 7 Calculated melting values for Tsl2 stem-loop and mutants

Supplementary Figure 8 Sequence for ATG5 exon 3 with U1 snRNP binding sites

Supplementary Figure 9 Protein analysis of enriched U1 snRNP

Supplementary Figure 10 Effect of compounds on the association of U1 snRNP to the 5’ss

Supplementary Figure 11 Proton NMR spectra for NVS-SM2 and NVS-SM4 with dsRNA

Supplementary Figure 12 Additional RNA binding data and computational SAR data

Supplementary Figure 13 1H and 13C NMR spectra for NVS-SM1





‐ S3 -

Supplementary Table 1. Small molecule screening data

Category Parameter Description

Assay Type of assay SMN – Luciferase Reporter assay

Target SMN

Primary measurement Luminescence measurement (Luciferase activity) at 0 and 24 hours after compound incubation

Key reagents Luciferin (Anaspec), Bright Glo (Promega)

Assay protocol Seed 2500 cells/well in 10 μL media in 1536-well plates using SelecT and

incubate O/N at 37C with 5% CO2

Aspirate the media down to 3.5 μL in each well by using AquaMax

Spin down for 1 minute using Vspin

Transfer 20 nL compounds/90% DMSO to assay plates from the compound source plates using Pintool (column 1 to column 44)

Dispense 0.5 μL/well of 4.05% DMSO into assay plates to column 47 and 48 with Flexdrop (Bottle C)

Dispense 0.5 μL/well of 72μM Resveratrol into assay plates to column 45 and 46 with Flexdrop (Bottle A)

Luciferin Add 0.5 μL/well of 270 μM Luciferin (final conc. 30μM) into all wells using Flexdrop

Vspin Spin down for 1 minute using Vspin

0hr Read on ViewLux

Incubation Incubate for 24 hours at 37C with 5% CO2

Library Library size ~1.4 million

Library composition Diversity set

Source Novartis compound library

Screen Format 1536-well

Concentration(s) tested 10 M

Plate controls Resveratrol, DMSO

Reagent / compound dispensing system Thermo HTS-3 automated system

Detection instrument and software Viewlux Imager

Assay validation/QC Average Z’ values and average RZ’ for the primary screen were 0.59 and 0.71, respectively

Correction factors n/a

Normalization n/a

Post-HTS analysis Hit criteria As active or superior to positive control, Resveratrol

Increases SMN Full reporter activity; Reduces SMN Reverse reporter activity

Hit rate < 1%

Additional assay(s) SMN ELISA

Confirmation of hit purity and structure LCMS; >80% of the compounds evaluated were comprised of the main compound (> 85% purity)


‐ S4 -

Supplementary Table 2. Candidate genes and exons identified in RNAseq

Gene symbol Entrez Gene ID Chromsome Start End Strand Exon number Type

ABHD10 55347 chr3 111709547 111709598 + 5 included

ADAM12 8038 chr10 127729913 127730804 - 19 included

AKT1 207 chr14 105259464 105259547 - 3 included

ANXA11 311 chr10 81940697 81940892 - 6 included

APLP2 334 chr11 129993507 129993674 + 9 included

APPL2 55198 chr12 105626869 105627183 - 2 included

ARMCX6 54470 chrX 100872273 100872360 - 3 included

ATG5 9474 chr6 106756239 106756366 - 3 skipped

AXIN1 8312 chr16 341190 341297 - 9 included

BAIAP2 10458 chr17 79084714 79084759 + 15 included

CCNB1IP1 57820 chr14 20785953 20786133 - 5 included

CCT7 10574 chr2 73462678 73462773 + 2 included

CEP57 9702 chr11 95528680 95528723 + 2 included

CSF1 1435 chr1 110468685 110469365 + 11 included

DLGAP4 22839 chr20 35127645 35127724 + 10 included

EPN1 29924 chr19 56187991 56188584 + 2 included

EPN1 29924 chr19 56188916 56189100 + 3 included

ERGIC3 51614 chr20 34142143 34142157 + 8 included

FOXM1 2305 chr12 2970465 2970578 - 11 included

GGCT 79017 chr7 30537357 30537512 - 4 included

GRAMD3 65983 chr5 125801986 125802028 + 6 included

HSD17B4 3295 chr5 118807331 118807395 + 4 included

LARP7 51574 chr4 113558800 113558943 + 3 included

LARP7 51574 chr4 113565581 113565644 + 4 included

LRRC42 115353 chr1 54413461 54413494 + 2 skipped

MADD 8567 chr11 47314094 47314147 + 24 included

MAN1B1 11253 chr9 139982720 139982850 + 3 included

MRPL39 54148 chr21 26960013 26960101 - 10 included

PCBP4 57060 chr3 51996826 51996908 - 3 included

PPHLN1 51535 chr12 42745687 42745851 + 4 included

PRKACB 5567 chr1 84609952 84610231 + 2 included

RAB23 51715 chr6 57086117 57086257 - 2 included

RAP1A 5906 chr1 112170092 112170185 + 2 included

RCC1 1104 chr1 28844745 28844820 + 6 skipped

SREK1 140890 chr5 65451893 65453568 + 4 included

SREK1 140890 chr5 65454637 65454760 + 5 included

STRN3 29966 chr14 31398407 31398517 - 8 included

STRN3 29966 chr14 31388172 31388312 - 9 included

TNRC6A 27327 chr16 24769660 24769681 + 4 skipped


a b

c

ParameterIV 1 mg/kg

PO 3 mg/kg

IV 1 mg/kg

PO 3 mg/kg

IV 1 mg/kg

PO 3 mg/kg

IV 1 mg/kg

PO 3 mg/kg

Cl (mL/min/kg) 22.7 220 20.5 21.6t1/2 (h) 8.5 9.8 10.5 12.2

Cmax (nM) 86 185 111 171

Tmax (h) 4.3 7 3 4

Vss (L/kg) 12.4 15.7 13.8 20.1

AUC0-24h (nM.h) 1788 1552 1615 3029 1704 1439 1477 2325

F (%) 29 63 29 53

Brain:Plasma @ 24h 1.5 1.4 1.8 1.3 1.1 0.7 1.8 1.4

NVS-SM1 NVS-SM2

mouse rat mouse rat

-10 -9 -8 -7 -6 -50

1

2

3

4

log[compound], M

NVS-SM3

NVS-SM1NVS-SM2

NVS-SM4SMN

pro

tein

(fold

from

DM

SO)

NVS-SM1NVS-SM2

-10 -9 -8 -7

1.0

1.5

2.0

SMN

pro

tein

(fold

from

DM

SO)

DMSOlog[compound], M

- S5 -

Supplementary Figure 1. In vitro activity and in vivo pharmacokinetics. (a) SMN ELISA data for SMN∆7 mouse myoblasts for all four compounds. (b) SMN protein ELISA in human fibroblasts for NVS-SM1 and NVS-SM2. (c) Pharmacokinetics (PK) table for NVS-SM1 and NVS-SM2. Intravenous dosing (IV) and oral dosing (PO) are shown. All data represent mean values ± s.e.m. of a minimum of two technical replicates representative of two biological replicates. Two-way analysis of variance (ANOVA) followed by Bonferroni test, protein at indicated doses vs. DMSO; *P<0.05, **P<0.01, ***P<0.001, ****P<0.0001.


a

4

0

1

2

3

Rel

ativ

e tra

nscr

ipt l

evel

s

SMNd7SMN_fl

0.5

1.5

2.5

3.5

*****

*

00.1

00.3

2 1.0 3.2 10 32 100

320

NVS-SM1 (nM)

0

1

2

0.5

1.5

2.5

**

*** ***

SMN

pro

tein

(fold

from

DM

SO)

00.1

00.3

2 1.0 3.2 10 32 100

320

NVS-SM1 (nM)

b c

NVS-SM1 (nM)

0 5 25 50

2.0

1.5

1.0

0.5

0.0

**

****

****Rel

ativ

e tra

nsci

rpt l

evel

s

SMNd7SMN_fl

- S6 -

Supplementary Figure 2. NVS-SM1 activity in additional cellular models. (a) qPCR data for SMN2 Δ7 (gray) and full-length transcript (black) in human iPS-derived neurons. (b) SMN protein ELISA in human iPS-derived neurons. (c) qPCR data for SMN2 Δ7 (gray) and full-length transcript (black) in SMA Type III patient PBMC. All data represent mean values ± s.e.m. of two technical replicates representative of two biological replicates. Two-way analysis of variance (ANOVA) followed by Bonferroni test. SMN2 Δ7 or SMN2_FL transcripts or SMN protein at indicated doses vs. DMSO; *P<0.05, **P<0.01, ***P<0.001; ****P<0.0001.


Age (days)

0 10 20 30 40 500

5

10

15

2033 days on-drug, 14 days off47 days on-drug

Treatmentbegins

Wei

ght (

g)

Holidaybegins

- S7 -

Supplementary Figure 3. Body weight analysis of NVS-SM1 dosing holiday in SMN∆7 mice. Cohorts of mice were treated with NVS-SM1 (1 mg/kg/d) from day 2 to 35, then one group (n=5) continued treatment to day 49 while the second (n=5) was treated with vehicle only. No changes in body weight were apparent following removal of NVS-SM1 treatment. This evaluation was performed once.


Junctions: NVS-SM1

Junctions: NVS-SM3

a b

Junctions: NVS-SM1

Junctions: NVS-SM3

1 2 3 4 5 6 7 8 9 10 11

1 2 3 4 5 6 7 8 9 10 11

log2

rel f

c

-20

2

log2

rel f

c

-20

2

log2

rel f

c

-1.0

0.5

2.0

log2

rel f

c

-1.0

0.5

2.0

1 2 3 4 5 6 7 8

1 2 3 4 5 6 7 8

Isof

orm

NM_004849Is

ofor

m NM_003502

NM_181050

AXIN1 ATG5

- S8 -

Supplementary Figure 4. Exemplar RNAseq exon transcript and junction variants. (a) Transcript isoforms for AXIN1 with exon junction reads. Two transcripts for AXIN1 are shown and attendant changes in exon junctions flanking exon 9 are shown. NVS-SM1 increases the exon 8-9 and exon 9-10 junctions (red), with a corresponding change in the exon 8-10 junction. No changes are apparent in NVS-SM3-treated (blue). (b) Transcript for ATG5 shown and attendant changes in exon junctions flanking exon 3 are shown. NVS-SM1 increases the exon 2-4 and 2-6 junctions, with an attendant decrease in the exon 2-3 and 3-4 junction reads (red). No changes in exon junctions are apparent in NVS-SM3-treated (blue). For both panels, changes shown are represented as log(2)-fold increases (up) or decreases (down) in junction reads, relative to DMSO.


CCT7

RQ

0

5

10

15

20****

Exclud

ed

Includ

ed

CEP57

0.0

0.5

1.0

1.5

RQ

****

**

Exclud

ed

Includ

ed

CSF1

0.0

0.5

1.0

1.5

2.0

2.5

RQ

**

Exclud

ed

Includ

ed

FOXM1

0

1

2

3

4

5

RQ

****

Exclud

ed

Includ

ed

MADD

0

20

40

60

80

RQ

****

Exclud

ed

Includ

ed

SMN2

0.0

0.5

1.0

1.5

2.0

2.5

RQ

****

****

Exclud

ed

Includ

ed

STRN3

0

5

10

15

RQ

****

*

Exclud

ed

Includ

ed

ADAM12R

Q

0.0

0.5

1.0

1.5

****

Exclud

ed

Includ

ed

APLP2

0

2

4

6

RQ

****

*

Exclud

ed

Includ

ed

ARMCX6

0

2

4

6

RQ

****

Exclud

ed

Includ

ed

BAIAP2

0.0

0.5

1.0

1.5

2.0

RQ

*

***

Exclud

ed

Includ

ed

- S9 -

Supplementary Figure 5. High-throughput qPCR validation of RNAseq splicing candidates. Assays for inclusion and exclusion of candidate exons were developed for 11 events identified from RNAseq. All exons as shown in Extended Data Table 1; STRN3 utilizes probeset for detection of exon 8 inclusion. DMSO treatment shown as black bars, and NVS-SM1 treatment shown as gray bars. RQ, relative quantitation to GusB, a housekeeping gene. Data represent mean values ± s.e.m. (n=8, from two independent experiments with four samples each). Subsequent analysis used two-way ANOVA, followed by Tukey post-hoc test (*P<0.05, **P<0.01, ***P<0.001, and ****P<0.0001). Data for SMN2 is shown as a control for NVS-SM1 activity.


a SMN1 E

Exon 6 Exon 7 Exon 8

SMN2E


BRCA - wild type E


BRCA - mutant E

Exon 19Exon 17 Exon 18

C

T

T

G SMN1SMN2

BRCA1 wt

BRCA1 mut

- Included- Skipped

b

0.001 0.01 0.1 10

20

40

60

80

100

NVS-SM2 (µM)

SMN2

BRCA1-MutBRCA1-WT

SMN1

Perc

enta

ge e

xon

incl

usio

n

c d

SMN1

SMN2

BRCA1 wt

BRCA1 mut

- Included- Skipped

- Included- Skipped

- Included

- Skipped

- Included- Skipped

- S10 -

Supplementary Figure 6. SMN2-BRCA minigene splicing data with NVS-SM2. (a) Schematic representation of SMN and BRCA1 minigene constructs – not shown to scale. Disease-causing mutations in SMN2 and BRCA1 are shown. (b) Representative RT-PCR data for constitutive splicing efficiencies for the constructs shown in (a). (c) RT-PCR data on compound-responsive changes in splicing efficiencies for constructs treated with NVS-SM2 (d) Graphical representation of compound responses for constructs. Data represent mean values ± s.e.m (n=3, representative of four independent experiments).


Tsl2 ∆G (kcal/mol)

Wild type

-1C

-2C

-15G

-16A

-5.66

-2.62

0.41

-1.28

-3.20

AA

UU

U

CC

GGAA

A U

AUU

CG

GA

U

U

AA

U

-1C-2C-15G

-16A

5´

3´Tsl2

C

- S11 -

Supplementary Figure 7. Calculated ∆G melting values for Tsl2 stem-loop and mutants. Schematic of Tsl2 stem-loop shown on left. Mutated nucleotides are shown with arrows, numbers represent distance from the 5’ss based on the minigene sequence.


5’UUGCUUUUGCCAAGAGUAAGUUAUUUGACGUUGGUAACUGACAAAGUGAAAAAGCACUUUCAGAAGGU

UAUGAGACAAGAAGACAUUAGUGAGAUAUGGUUUGAAUAUGAAUUCACACCACUGAAAUGgugaguga3’

MaxEnt = 9.35

MaxEnt = 10.13

E


U1 binding site

5´ ss

- S12 -

Supplementary Figure 8. Sequence for ATG5 exon 3 with U1 snRNP binding sites. A schematic of ATG5 illustrating the cryptic U1 binding site within exon 3 is shown above the sequence. Within the sequence, U1 sites are underlined, with the predicted U1 cryptic recognition site in red, native 5’ss in blue, exon sequences in uppercase, and intron sequences in lowercase. MaxEnt scores for predicted U1 binding shown by the sequences for cryptic and native 5’ss.


a

U5

U5

U1-70k

U1AB, B´

U1CD1, D2, D3

EF, G

260

16011080

6050

40

30

2015

10

20

15

10

37

50

100

U1A

U1C

150

250

kDa

20

15

10

37

50

100

U1-70k

U1C

150

250

bkDa kDa

- S13 -

Supplementary Figure 9. Protein analysis of enriched U1 snRNP. (a) SDS-PAGE of U1 snRNP, showing all expected U1 specific proteins as well as the Sm proteins. (b) Western blots showing detection of U1A, U1C and U1-70k proteins by their respective antibodies at their expected molecular weights using 2-color fluorescent detection. kDa, kilodaltons.


400

300

200

100

0

Time (s)

Res

pons

e (R

U)

U1 snRNP with DMSO

NVS-SM2 alone

50 100 150 200

U1 snRNP with NVS-SM2

400

300

200

100

0

400

200

0

600

Time (s) Time (s)

Res

pons

e un

its (R

U)


U1 snRNP with DMSO


U1 snRNP with DMSO

NVS-SM2 alone NVS-SM4 alone

50 100 150 200 50 100 150 200

- S14 -

Res

pons

e (R

U)

Supplementary Figure 10. Effect of compounds on the association of U1 snRNP to the 5’ splice site. Effect of compounds (10 µM) in association phase only on binding of U1 snRNP to 5’ss SMN RNA as measured by surface plasmon resonance. Representative sensorgrams are shown (from a minimum of two independent experiments, all injections containing U1 snRNP were performed in triplicate), with DMSO control shown in fuchsia, compound treatment shown in blue, and compound alone shown in orange. RU, resonance units.


NVS-SM4 + dsRNANVS-SM4

NVS-SM2 + dsRNANVS-SM2

- S15 -

Supplementary Figure 11. Proton NMR spectra for NVS-SM2 and NVS-SM4 with dsRNA. Proton NMR spectra for NVS-SM2 (top panel) and NVS-SM4 (bottom panel) in the absence (red) or presence (black) of RNA 11-12 duplex. NVS-SM2 spectral peaks show downfield shift and peak broadening in the presence of dsRNA.


a

0 100 500400200 300

20

40

60

80

100

120

140

0

Res

pons

e (R

U)

Time (s)

-30

-25

-20

-15

Compound

NVS-SM1

NVS-SM2

NVS-SM3

NVS-SM4

VlsS

core

(kca

l/mol

)

b

Unf

avor

able

Favo

rabl

e

RNA with 10 µM NVS-SM2RNA with 1 µM NVS-SM2RNA only

- S16 -

Supplementary Figure 12. Additional RNA binding data and computational SAR data. (a) Surface plasmon resonance data for effect of NVS-SM2 on the binding of U1 snRNA to SMN 5’ss RNA. Coinjections of U1 snRNP with NVS-SM2 are shown at 1 µM (blue) and 10 µM (green) compound along with the DMSO alone control (fuchsia). Representative sensorgrams are shown from two independent experiments. RU, resonance units. (b) VLS docking score (kcal / mol) versus compound ID after selection of the optimal small molecule-RNA conformation complex.


- S17 -

Supplementary Figure 13. 1H and 13C NMR spectra for NVS-SM1.

-4-3-2-1012345678910111213141516f1 (ppm)

12.1

72.

122.

11

1.12

2.33

1.20

1.12

2.11

1.09

0.96

0.93

0.00

1.06

1.54

1.55

1.85

1.87

1.88

1.88

1.91

2.29

2.30

2.32

2.33

2.51

DM

SO2.

51 D

MSO

2.52

DM

SO2.

52 D

MSO

2.53

DM

SO

5.67

5.69

5.70

5.71

5.72

7.25

7.26

7.28

7.28

7.29

7.30

7.49

7.51

7.94

7.96

8.23

8.50

8.52

8.67

8.70

9.50

9.53

0102030405060708090100110120130140150160170180190f1 (ppm)

24.9

5

29.0

938

.72

38.9

3 DM

SO39

.08

DMSO

39.1

4 DM

SO39

.35

DMSO

39.5

6 DM

SO39

.77

DMSO

39.8

1 DM

SO39

.97

DMSO

56.5

6

67.9

9

113.

3611

5.04

116.

3112

0.27

120.

40

128.

0412

9.07

131.

26

136.

02

156.

0315

8.34

162.

28

1H NMR

13C NMR

N

HN

NN

O

NH

OH

N

HN

NN

O

NH

OH



- S18 -

1H NMR

13C NMR

-4-3-2-1012345678910111213141516f1 (ppm)

1

6.00

5.99

4.42

3.13

0.97

2.13

1.05

1.09

1.40

0.59

0.94

0.00

1.10

1.27

1.42

1.45

1.48

1.52

1.53

1.55

1.56

2.49

DM

SO2.

50 D

MSO

2.50

DM

SO2.

51 D

MSO

2.51

DM

SO2.

953.

33 H

DO

4.92

4.95

4.98

7.17

7.18

7.19

7.20

7.21

7.21

7.34

7.36

7.82

7.84

8.12

8.20

8.22

12.9

8

13.8

2

-100102030405060708090100110120130140150160170180190200210f1 (ppm)

28.5

128

.97

34.4

6

40.5

5

47.3

6

113.

4011

4.94

115.

2911

5.96

120.

6312

5.21

126.

27

134.

86

151.

04

157.

6115

8.49

N

HN

NN

N

NH

OH

N

HN

NN

N

NH

OH



- S19 -

1H NMR

13C NMR

-30-20-100102030405060708090100110120130140150160170180190200210220230240f1 (ppm)

27.9

129

.74

34.1

9

41.8

4

51.3

153

.06

116.

0811

9.29

119.

4412

0.71

126.

6612

6.71

128.

7512

9.30

131.

0713

8.40

152.

34

159.

4515

9.49

8.5 8.0 7.5 7.0 6.5 6.0 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.0 1.5 1.0Chemical Shift (ppm)

0

0.25

0.50

0.75

1.00

Nor

mal

ized

Inte

nsity

6.006.064.133.040.952.031.910.930.990.991.000.941.01

Methanol

M06(t)M03(d)

M02(s) M08(s)

M13(s)

M14(s)

M05(dd)

M04(d)

M01(d)

M07(m)

Methanol

M11(m)

M09(m)

M10(s)

M12(m)

1.251.40

1.59

1.621.68

1.69

1.72

1.72

3.02

5.20

6.966.986.99

7.00

7.167.

167.

167.

207.30

7.33

7.74

7.757.78

8.08

8.10

NN

N

NH

OH

N

N

NN

N

NH

OH

N

N



- S20 -

1H NMR

13C NMR

-0.50.00.51.01.52.02.53.03.54.04.55.05.56.06.57.07.58.08.59.09.5f1 (ppm)

5.89

5.92

2.12

2.05

2.94

1.01

1.01

1.01

1.00

2.05

0.00

1.24

1.38

1.54

1.57

1.60

1.66

1.67

1.70

1.71

2.95

3.30

MeO

D3.

31 M

eOD

3.31

MeO

D3.

32 M

eOD

3.32

MeO

D

4.89

HDO

5.18

5.20

5.21

5.22

5.24

6.56

6.57

6.60

6.60

6.68

6.69

6.71

6.71

7.08

7.11

7.60

7.62

7.63

7.64

7.66

7.66

20253035404550556065707580859095100105110115120125130135140145150155160165170f1 (ppm)

2.07

1.99

1.93

0.88

1.96

1.98

0.84

0.89

0.41

1.00

0.42

27.7

129

.69

33.9

2

41.6

8

48.3

3

53.7

0

104.

2310

4.47

113.

6811

3.71

113.

9511

6.32

116.

44

130.

2613

0.33

131.

4913

1.54

148.

8814

8.90

159.

7016

1.17

162.

5416

2.66

163.

63

NN

N

NH

FHO

NN

N

NH

FHO


SUPPLEMENTARY INFORMATION SMN2 Splice Modulators Enhance ... · SUPPLEMENTARY INFORMATION SMN2...

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