Arrowhead Pharmaceuticals : 2024 Muscular Dystrophy Association (MDA) Clinical & Scientific Conference View Presentation

Silencing DMPK gene by ARO-DM1, an

RNAi therapeutic, for Type 1 Myotonic Dystrophy

Jonathan Van Dyke, Teng Ai, Xiaokai Li, Adijan Kuckovic, Daniel Braas, Holly Hamilton, Maria Afrazi,

Tao Pei, James Hamilton, Zhi-Ming Ding. Arrowhead Pharmaceuticals, Madison, WI

Overview:

• Pathogenesis of type I myotonic dystrophy (DM1) is driven by an expanded CUG trinucleotide repeat in the 3'- untranslated region of DMPK transcripts.
• Pathogenic transcripts of human DMPK sequester RNA splicing factors and thereby cause leading to myotonia, muscular dystrophy, cataracts, and cardiac conduction abnormalities.
• There is no approved drug for treating the root cause of DM1.
• To develop a therapeutic for DM1, we designed siRNA conjugates to silence DMPK mRNA in skeletal muscles
• ARO-DM1and S-ARO-DM1 were identified as the best 2 conjugates, which target different positions of DMPK genes.
• In NHPs, DMPK mRNA expression in quadriceps and triceps was substantially decreased after two IV doses of ARO- DM1 or S-ARO-DM1, respectively.
• In the TREDT960i/HSA-rtTA mouse model of DM1 harboring a pathogenic DMPK transgene (DMPK-CUG),S-ARO-DM1:
• • Decreased the DMPK-CUG expression
  • Corrected the spliceopathies caused by overexpression of DMPK-CUP transgene

When intravenously injected weekly at 20 and 40 mpk respectively.

Pharmacodynamic study of ARO-DM1 and S-ARO-DM1 in cynomolgus

monkeys

PD

						• After a single dose, muscular DMPK expression
RNAi,					was reduced in quadriceps and triceps.
13.3 mpk, IV					Knockdown was maintained through Day 85 after
Biopsy					a second dose on Day 29.
						• ARO-DM1 and S-ARO-DM1 exhibited similar
						potency in quadriceps; however, in triceps, S-
N =3					ARO-DM1 was slightly less potent.

Pharmacological study of S-ARO-DM1 in TREDT960i/HSA-rtTA mouse model of DM1l	Conclusions
TREDT960i/HSA-rtTA mouse model transgene	+ Doxycycline	Human DMPK-CUG		•	ARO-DM1 and S-ARO-DM1 are
	TRE		960 CTG repeats + HSA promotor rtTA	Mouse Dmpk
	Human DMPK			potent RNAi therapeutics that
	(e11-15 and 3' flanking genomic)					silence skeletal muscle DMPK
										mRNA
									•	S-ARO-DM1 exhibited efficacies
										in reducing the expression of
										pathogenic DMPK-CUG
										transgene and correcting
										spliceopathies in the skeletal
										muscles of a mouse model of
							• Using RNAscope, human	• S-ARO-DM1 was selected for		DM1
• DMPK-CUG expressing mice and noncarrier	DMPK-CUG transcripts were	pharmacological studies due to its sequence	•	ARO-DM1 phase 1/2a studies
complementarity to DMPK-CUG in the
control mice (NCAR) were administered	detected in the myonuclei		are in progress in patients with
transgenic mice.
doxycycline by diet over 50 days.		while endogenous mouse		DM1
• In DMPK-CUG expressing mice, DMPK-CUG	Dmpk was detected both in	• Weekly administration of S-ARO-DM1
expression was induced in skeletal muscles.	the myonuclei as well as in	decreased DMPK-CUG expression induced
• DMPK-CUG expression plateaued by Day 22.	sarcoplasm.	by doxycycline.

Analysis of Spliceopathies in the TREDT960i/HSA-rTA mice treated with S-ARO-DM1

Relative Mis-splicing by ddPCR-based Competitive Probe Analysis

ddPCR-based Competitive Missplicing Assay
			Inclusion
		Exon A	Exon B	Exon C
			Exclusion
Inclusion Detected		FAM		Exclusion Detected	HEX
	HEX			FAM
Fwd Primer				Fwd Primer
Exon A	Exon B	Exon C		Exon A	Exon C
		Rev Primer			Rev Primer

Mis-splicing Analysis by RNAseq

*

†

NCAR, 52.9mpk S-ARO-DM1, +Dox

DMPK-CUG, Saline, -Dox

DMPK-CUG, Saline, +Dox

DMPK-CUG, 26.4mpk S-ARO-DM1, +Dox

DMPK-CUG, 52.9mpk S-ARO-DM1, +Dox

Female

Male

Targeted Gene Panel

Abcc9*	Eya4*	Mbnl2	Rapgef1*†
Atp2a1*†	Fbxo31*	Mpdz	Ryr1*†
Best3	Fn1	Neb	Slc8a3
Bin1*†	Jag2	Nfix*	Tnik*
Cacna2d1*†	Kif13a*	Opa1	Trappc9*
Clasp1	Ldb3*†	Phactr4*	Trim55*†
Clcn1†	Lrrfip2*	Phka1	Ttn
Cpeb2	Map3k4*	Ppp1r12b*	Vps39*
Dctn4	Mbnl1*	Ppp3cc*

• Spliceopathies were analyzed by 2 different assays using the mRNA from the gastrocnemius of DMPK- CUG and NCAR mice: ddPCR-based competitive mis-splicing and RNAseq analysis.
• Mis-splicingof 3 marker genes reported in the literature was detected by the ddPCR based assay.
• S-ARO-DM1treatment corrected the mis-splicing of those 3 marker genes.
• Mis-splicingof a targeted 35 gene panel was examined by RNAseq.
• Composite Percent Splice In Index (PSI)
  was analyzed for the entire panel; the 20 most responsive genes to S-ARO-DM1, and 8 genes primarily expressed in skeletal muscles.
• S-ARO-DM1treatment corrected the mis-splicing in all 3 panels.