Sarepta Therapeutics : Wu et al. A Peptide-conj PMO Incr Exon Skip Dystr Prod & Grip Stren in Mouse Mod of DMD

OTS 2020

SRP-5051, a Peptide-conjugated PMO, Increases

Exon Skipping, Dystrophin Production, and Grip Strength in a Humanized Mouse Model of Duchenne Muscular Dystrophy

Leslie C.L. Wu, John R. Hadcock, Miralem Prijic, Jenna Wood, Kamela Bellovoda, Bryan Mastis, Jianbo Zhang, Sam Foley, Mohammad Shadid, Claire Mukashyaka, Shawn Harriman, Annika B. Malmberg

Presented by Leslie C.L. Wu

Sarepta Therapeutics, Inc., Cambridge, MA, USA

Disclosures

• All authors are employees of Sarepta Therapeutics, Inc. and may own stock in the company
• The study was funded by Sarepta Therapeutics, Inc.
• Editorial support was provided by Eloquent Scientific Solutions and was funded by Sarepta Therapeutics, Inc.
• Products are investigational only

2

Introduction

• Duchenne muscular dystrophy (DMD) is a severe, X-linked neuromuscular disease caused by mutations in the dystrophin gene1
• • Dystrophin mutations leading to deletions flanking exon 51 account for 13% of all DMD patients2
• 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 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 level7,8
• SRP-5051is an investigational PPMO designed to skip exon 51 of the DMD

Objective: To evaluate the biological and functional efficacy of SRP-5051 in a humanized mouse model of DMD

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.

3

Humanized DMD Transgenic Mouse Allows Assessment of SRP-5051 Pharmacology in a Preclinical Disease-Relevant Model of DMD

hDMDdel52/mdx mice

mouse

49

50

51

53

54

human DMD transgene with exon 52 deleted by gene editing is inserted into mdx mouse genome, results in out of frame DMD transcript and lack of dystrophin protein expression

Treatment with SRP-5051

hDMD del52/mdx

				Dystrophin
49	50	53	54	ɑ-actinin

SRP-5051 induced exon 51 skipping, which results in restoration of DMD reading frame, and expression of truncated, but functional dystrophin protein

Experimental design summary:

• Single injection dose response and time course: exon skipping and dystrophin were measured
• Repeated doses time course: exon skipping, dystrophin and grip strength were measured

4

Single-doseSRP-5051 Increased Exon Skipping and Dystrophin Protein in hDMDdel52/mdx Mice

Dose response (single dose): 6-8 w/o male hDMDdel52/mdx mice were injected with SRP-5051 IV at indicated doses and analyzed 7 post dose

Dystrophin Level (% WT) Exon 51 skipping (%)

HEART

100			***					100
80			****						80
				**
60													60
40																				40
20																				20
0																					0

15	*	25
	20
10
	15
5		10
	5
0		0

BICEPS

							****						100
												80
											***
															60
															40
															20
																0

														**		25
																		20
																		15
																		10
																		5
																			0

QUADRICEPS

***

Biological Activity at the 100 mg/kg Dose

Exon

Dystrophin

skipping

protein

Heart

24%

5%

*

**

Skeletal

22%

10%

muscles

Each bar represents mean ± SE. One-way ANOVA was used to compare means among dose groups. Exon skipping results showed significant difference among the groups (P<0.0001), whereas dystrophin level showed significant differences among groups in heart (P=0.0042), biceps (P=0.0007) and quadriceps (P=0.0047). Dunnett multiple comparison test was used to compare the 2 groups of interest; *, **, ***, **** denotes P< 0.05, 0.005, 0.0005 or 0.0001, respectively.

5

Exon Skipping was Sustained 4 Weeks After Single Dose SRP-5051 and Remained Detectable to 12 Weeks in hDMDdel52/mdx Mice

Time course (single dose): 6-8 w/o male hDMDdel52/mdx mice were injected with SRP-5051 at 100 mg/kg IV; exon skipping and dystrophin were measured 4, 8, and 12 weeks post-dose

HEART

BICEPS

QUADRICEPS

Exon 51 skipping (%)

100

80

60

40

20

0

4

8

12

Time Post Injection (weeks)

(%)	100	***		(%)	100
80		80
skipping		*	skipping
60		60
40			40
51			51
Exon	20			Exon	20
	0				0

4

8

12

Time Post Injection (weeks)

	(% WT)	150
*	100
	level
	Dystrophin	50
	0

4

8

12

Time Post Injection (weeks)

4

8

12

Time Post Injection (weeks)

• Dystrophin was undetectable in the vehicle-treated samples
• Exon skipping and dystrophin protein level were maintained for ≥8 weeks after a single dose

Each bar represents mean ± SD. One-way ANOVA was used to compare group means across 3 time points (biceps, P=0.0002; exon skipping for quadriceps, P=0.0443). Dunnett multiple comparison test was used to compare the 2 timepoints of interest; *, *** denotes P<0.05 or 0.0005, respectively.

6

Every 4 Weeks Repeated SRP-5051 Dosing Resulted in Increased Exon Skipping and Accumulation of Dystrophin in hDMDdel52/mdx Mice

Time course (repeated doses): 6-8 w/o male hDMDdel52/mdx mice were injected with SRP-5051 100 mg/kg IV every 4 weeks and analyzed 4 weeks after 1 dose, 3 doses, or 5 doses

Dystrophin Level (% WT) Exon 51 skipping (%)

		HEART			QUADRICEPS		DIAPHRAGM
100				100				100
80	****			80	****			80	****		• Exon skipping reached a
60				60				60
40				40				40				steady-state after 3 doses
20				20				20
									(12 weeks)
0	4	12	20	0	4	12	20	0	4	12	20
	Time Post 1st injection (weeks)		Time Post 1st injection (weeks)		Time Post 1st injection (weeks)
			*				*			**	•	Dystrophin protein level
100		****		100		****		150		****		continued to increase up
80		***		80		**				***		to 5 doses (20 weeks)
60				60				100
40				40				50
20				20
0	4	12	20	0	4	12	20	0	4	12	20
	Time Post 1st injection (weeks)		Time Post 1st injection (weeks)		Time Post 1st injection (weeks)

Each bar represents mean ± SEM. One-way ANOVA was used to compare group means (P<0.0001 for exon skipping and dystrophin level in all structures examined). Dunnett multiple comparison test was used to compare the 2 groups of interest; *, **, ***, **** denotes P< 0.05, 0.005, 0.0005 or 0.0001, respectively.

7

Single and Repeated SRP-5051 Dosing Every 4 Weeks Restored Grip Strength in hDMDdel52/mdx Mice to Wild Type Levels

Time course (repeated doses): hDMDdel52/mdx mice were injected with SRP-5051 100 mg/kg IV every 4 weeks and analyzed every 4 weeks

Normalized Force (force g/weight g)

GRIP STRENGTH

8

P<0.0001

P<0.0001

P<0.0001

P=0.0003

P<0.0001

6

4

2
0	N/A
Week 0	Week 4	Week 8	Week 12	Week 16	Week 20
		(single dose)

Unpaired T-test was used to compare the grip strength between the 2 groups at each timepoint.

Saline 100mg/kg

8

Conclusions

• This is the first-time examination of the pharmacology of SRP-5051, a peptide conjugated PMO, in a disease- relevant, humanized mouse model of DMD
• A single dose of SRP-5051 resulted in sustained exon 51 skipping and human dystrophin levels in muscle and heart, which was associated with improved grip strength, a surrogate measure of muscle function
• • Significant exon 51 skipping and human dystrophin were detected in skeletal muscle and heart 7 days post injection of ascending doses and for at least 8 weeks after a single 100 mg/kg dose
• Once every 4-weekSRP-5051 dosing for 20 weeks resulted in improved pharmacodynamic effect compared with vehicle-treated animals
• • Improvement in grip strength was sustained throughout the 20-week study
• These studies further support the clinical investigation of SRP-5051
• • A phase 2 multiple-ascending-dose study is currently ongoing (NCT04004065)

9

Questions?

Please direct any questions you may have to the Sarepta Medical Information team at

[e-mail]

10