Sarepta Therapeutics : P.6 Hadcock et al. Biological Efficacy of PPMO SRP-5051 in Preclin. Models of DMD

Presented at the 2021 Muscular Dystrophy Association Virtual Clinical & Scientific Conference, March 15−18, 2021 | Corresponding author: John R. Hadcock;[e-mail]

Biological Efficacy of the Peptide-Conjugated Phosphorodiamidate Morpholino Oligomer SRP-5051 in Preclinical Models of Duchenne Muscular Dystrophy

John R. Hadcock,1 Peter M. Burch,1 Amy Erickson,1 Sam Foley,1 Brian Mastis,1 Pavlo Kovalenko,1 Blandine Mille-Baker,2 Marie Claire Mukashyaka,1

Miralem Prijic,1 Mohammad Shadid,1 Fabian Stavenuiter,2 Jenna Wood,1 Leslie C.L. Wu,1 Mark Wysk,1 Jianbo Zhang,1 Annika B. Malmberg,1 Shawn Harriman1

1Sarepta Therapeutics, Inc., Cambridge, MA, USA; 2Charles River Laboratories, Leiden, Netherlands

OObbjejectcitvivee:

CONCLUSIONS

TTooininvvesetsigtaigteattheetbhieolobgioiclaol gefifciacalcy of the peptide-conjugated efficacy of SRP-5051 in phosphorodiamidate morpholino oligomer (PPMO) SRpPr-e5c0l5in1,icdaelsimgnoedetolsskip exon 51 of the DMD gene, in preclinical models

• • Exon skipping and dystrophin production were demonstrated in Duchenne muscular dystrophy (DMD) patient myotubes in vitro at cellular SRP-5051 concentrations of 100−1000 nM

  Key Takeaways:

  • • Dose-dependent efficacy of SRP-5051 was

    Key Takeaways

  • • dDeomseo-ndsetpreanteddenint eaflflicparceycloinf iScRaPl -m50o5d1ewlsas demonstrated in myotubes, mice, and

• • In hDMDdel52/mdx mice, a single SRP-5051 injection resulted in dose-dependent increases in exon skipping and human dystrophin protein - Repeated dosing every 4 weeks improved pharmacodynamic effects, with dystrophin protein accumulation through 20 weeks (5 doses) and sustained grip strength improvement through 20 weeks

  • • nRoenpheuamtamn opnritmhlaytedporseinclginiimcapl rmovoeddels pharmacodynamic effects and grip

    • • These data justify the SRP-5051 strength in hDMD del52/mdx mice dosing regimen (every 4 weeks)

• • In nonhuman primates, a single SRP-5051 infusion resulted in a dose-dependent increase in exon skipping and accumulation of drug in muscle for up to 28 days - Repeated dosing every 4 weeks over 12 weeks showed exon skipping increased with each infusion, and SRP-5051 appeared to be well tolerated

  • • uSRsePd-5in05o1ngwoainsgwcelinllictoalesrtautdeieds andinsuhpepaoltrht yfuprrthimeractelins ical investigation

• • SRP-5051 showed dose-dependent efficacy in preclinical models, justifying the SRP-5051 dosing regimen (every 4 weeks) used in ongoing clinical studies and supporting further clinical investigation of this PPMO

ACKNOWLEDGMENTS & DISCLOSURES

Editorial support was provided by Kristin M. Allan, PhD, of Eloquent Scientific Solutions, and was funded by Sarepta Therapeutics, Inc. Disclosures: JRH, PMB, AE, SF, PK, MCM, MP, MS, JW, LCLW, JZ, ABM, and SH are employees of Sarepta Therapeutics, Inc., and may own stock in the company. BM and MW were Sarepta employees at the time of the analysis. BM-B and FS are employees of Charles River Laboratories and may own stock in the company. Products are investigational only.

Exon51skipping(%)

0 10 30 100

Dystrophinlevel (%ofh DMD / mdx mice)

Exon skipping and dystrophin protein increased after a single dose of SRP-5051

0 10 30 100

SRP-5051 dose (mg/kg)

SRP-5051 dose (mg/kg)Exon51skipping(%)

4

8

12

Dystrophinlevel (%ofh DMD / mdx mice)

4 8 12

Time post injection (weeks)

Time post injection (weeks)

Exon skipping and dystrophin protein levels were sustained 4 weeks after single-dose SRP-5051 and remained detectable to 12 weeks

*

Exon51skipping(%)Dystrophinlevel (%ofh DMD / mdx mice)

****

SRP-5051 dosing every 4 weeks resulted in increased exon skipping and accumulation of dystrophin

4

12

20

4

12

20

Time post 1st injection (weeks)

Time post 1st injection (weeks)

Results for quadriceps are shown. Each bar represents mean ± SE (single dose and repeated dosing) or ± SD (single-dose time course). 1-way analysis of variance was used to compare means among dose groups. Dunnett multiple comparison test was used to compare the 2 groups of interest: *P<0.05, **P<0.005, ***P<0.0005, **** P<0.0001.

BACKGROUND

• • Duchenne muscular dystrophy (DMD) is a severe, X-linked neuromuscular disease caused by mutations in the dystrophin (DMD) gene1 ―Dystrophin mutations leading to deletions flanking exon 51 account for 13% of all patients with Duchenne muscular dystrophy (DMD)2

• • Phosphorodiamidate morpholino oligomers (PMOs) are an effective treatment approach for patients with DMD3-6

• • PMOs are designed for targeted skipping of exons within the DMD gene; they restore the reading frame and allow for production of an internally truncated but functional dystrophin protein

• • Peptide-conjugated PMOs (PPMOs) are a next-generation chemistry platform in which a cell-penetrating peptide is conjugated to the PMO backbone, with the goal of increasing cellular uptake, exon skipping, and dystrophin production7,8

• • SRP-5051 is an investigational PPMO designed to skip exon 51 of the DMD gene

Patient-derived myotube model

• • Immortalized myoblasts derived from the paravertebral muscles of a healthy male donor and a male patient with DMD with an exon 52 deletion (DMD del52)

  ― Human telomerase and cyclin-dependent kinase 4 (CDK4)-immortalized myoblasts were provided to Sarepta by the Association Institut de

Myologie (Paris, France); both donors were aged 16 years

• • For DMD del52 cellular assays, myoblasts were allowed to differentiate for 2 days before treatment with PPMO (SRP-5051 or RC-1001) for 4 days

  • ― RC-1001: negative control PPMO, with no homology to the human DMD gene

  • ― Exon skipping was measured by droplet digital PCR (ddPCR)

  • ― Dystrophin protein production was measured through imaging analysis

• • To directly measure SRP-5051 cellular concentration by liquid chromatography-mass spectrometry, myoblasts were first allowed to differentiate for 3 days before treatment with PPMO (SRP-5051 or RC-1001) for 3 days, and washed ― Exon skipping was measured by ddPCR ― Dystrophin protein was measured by capillary Western

hDMDdel52/mdx mouse model

• • Human DMD transgene with exon 52 deleted by gene editing was inserted into the mdx mouse genome, resulting in an out-of-frame human DMD transcript and lack of dystrophin protein expression

• • 6-8-week-old male hDMDdel52/mdx mice were used for experiments

• • Single dose ― Dose response: SRP-5051 IV at 10, 30, or 100 mg/kg; analyzed 7 days post dose for exon skipping and dystrophin ― Time course: SRP-5051 IV at 100 mg/kg; analyzed 4, 8, and 12 weeks post-dose for exon skipping and dystrophin

• • Repeated doses ― SRP-5051 IV at 100 mg/kg every 4 weeks

  • • Analyzed after 1, 3, or 5 doses for exon skipping and dystrophin

  • • Analyzed every 4 weeks for grip strength

Nondystrophic nonhuman primate model

• • Healthy male cynomolgus monkeys received a single 1-hour IV infusion of SRP-5051 at dose levels of 30 and 60 mg/kg ― Blood samples were collected on Day 1: predose and 1, 2, 4, 8, 12, 16, and 24 hours post infusion ― Muscle samples were collected on Days 2, 5 (only for the 30-mg/kg group), 10, 15, 22, and 28 by biopsy

• • For repeated dosing, animals received 1-hour IV infusion of vehicle or SRP-5051 30 mg/kg or 60 mg/kg once every 4 weeks ― Muscle biopsy tissue was collected on the second day after each infusion

REFERENCES

Presented at the 2021 Muscular Dystrophy Association Virtual Clinical & Scientific Conference, March 15−18, 2021 Corresponding author: John R. Hadcock;[e-mail]

1. Birnkrant DJ, et al. Lancet Neurol. 2018;17:251-67. 2. Aartsma-Rus A, et al. Hum Mutat. 2009;30:293-9. 3. Popplewell LJ, et al. Mol Ther. 2009;17:554-61.

4. Exondys 51 [package insert]. Cambridge, MA: Sarepta Therapeutics, Inc.; 2020. 5. Vyondys 53 [package insert]. Cambridge, MA: Sarepta Therapeutics, Inc.; 2020. 6. Viltepso [package insert]. Paramus, NJ: NS Pharma, Inc.; 2020. 7. Gan L, et al. Poster presented at the 2019 Muscular Dystrophy Association (MDA) conference. April 13-17, 2019, Orlando, FL. 8. Echevarría L, et al. Hum Mol Genet. 2018;27:R163-72.