Wave Life Sciences : Chemical Design of Oligonucleotides That Support Targeted RNA Editing in the CNS of Non-Human Primates

Potent, Durable mRNA Knockdown in Extrahepatic Tissues Using siRNAs With Novel Phosphoryl Guanidine Backbone Variants

Wei Liu, Principal Scientist, Biology

May 10, 2024

Presented at the American Society for Gene & Cell Therapy 27th Annual Meeting

Forward-looking statements

This document contains forward-looking statements. All statements other than statements of historical facts contained in this document, including statements regarding possible or assumed future results of operations, preclinical and clinical studies, business strategies, research and development plans, collaborations and partnerships, regulatory activities and timing thereof, competitive position, potential growth opportunities, use of proceeds and the effects of competition are forward-looking statements. These statements involve known and unknown risks, uncertainties and other important factors that may cause the actual results, performance or achievements of Wave Life Sciences Ltd. (the "Company") to be materially different from any future results, performance or achievements expressed or implied by the forward-looking statements. In some cases, you can identify forward- looking statements by terms such as "may," "will," "should," "expect," "plan," "aim," "anticipate," "could," "intend," "target," "project," "contemplate," "believe," "estimate," "predict," "potential" or "continue" or the negative of these terms or other similar expressions. The forward-looking statements in this presentation are only predictions. The Company has based these forward-looking statements largely on its current expectations and projections about future events and financial trends that it believes may affect the Company's business, financial condition and results of operations. These forward-looking statements speak only as of the date of this presentation and are subject to a number of risks, uncertainties and assumptions, including those listed under Risk Factors in the Company's Form 10-K and other filings with the SEC, some of which cannot be predicted or quantified and some of which are beyond the Company's control. The events and circumstances reflected in the Company's forward-looking statements may not be achieved or occur, and actual results could differ materially from those projected in the forward-looking statements. Moreover, the Company operates in a dynamic industry and economy. New risk factors and uncertainties may emerge from time to time, and it is not possible for management to predict all risk factors and uncertainties that the Company may face. Except as required by applicable law, the Company does not plan to publicly update or revise any forward-looking statements contained herein, whether as a result of any new information, future events, changed circumstances or otherwise.

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Disclosures

Wei Liu, Naoki Iwamoto, Subramanian Marappan, Himali Shah, Snehlata Tripathi, Erin Purcell-Estabrook, Khoa Luu, Anthony Lamattina, Qianli Pan, Fangjun Liu, Frank Favaloro, Arindom Chatterjee, Tomomi Kawamoto, Genliang Lu, Jake Metterville, Priyanka Shiva Prakasha, Hailin Yang, Yuan Yin, Lola Owen, Hui Yu, Michael Byrne, Pachamuthu Kandasamy, Chandra Vargeese

• All authors are employees of Wave Life Sciences

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Oligonucleotide-directed gene silencing by RNA interference

siRNA induce mRNA degradation	GalNAc conjugation mediates hepatocyte
delivery of siRNA1
	Endogenous AGO2	mRNA
		(liver)
RISC	siRNA

Extrahepatic tissue targeting remains a major challenge for the field

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1Zhang L, Liang Y, Liang G, Tian Z, Zhang Y, Liu Z and Ji X (2022) Front. Pharmacol. 13:1090237. doi: 10.3389/fphar.2022.1090237. GalNac: N-Acetylgalactosamine

Wave's ability to rationally design oligonucleotides enables access to unique disease targets

B	Base			O 5'	O B
	X	3'O	R 2'
			-
R	2'-Ribose		O	P	5' O
X	Stereochemistry and		O	B
	backbone modification			3'O	R 2'
Phosphoryl guanidine (PN-1)	Phosphorothioate (PS)	Phosphodiester (PO)

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Incorporation of PN chemistry improves GalNAc-siRNA potency and durability in mice

Ttr mRNA in liver (C57BL/6J mice)

Ago2 loading in liver

(C57BL/6J mice)

25	*

Serum Ttr protein (C57BL/6J mice)

% mRNA remaining (Ttr/Hprt - Relative to PBS)

125

100

75

50

25

0

• **

Relativefold change	20
15
****
10
guide/miRTtr122-
	5
	0

	150
Mean serum Ttr (±sem) Relative to pre-serum	100
50
	0

PBS 0.6 2.0 6.0

Dose (mg/kg)

10	20	30	40
	Time (days)

PBS	PS/PO backbone	PS/PO/PN-1 backbone (non-optimalPN-1)
Reference compound	PS/PO/PN-1 backbone

Left, C57BL/6J mice were treated with 0.6, 2, or 6 mg/kg of the indicated siRNA or PBS by SC injection on day 0. Liver Ttr mRNA levels were quantified by RT-PCR1-week later. Stats: 2-way ANOVA ** P<0.01, **** P<0.0001. Middle, Ago2 loading was quantified by RT PCR from livers in panel A after 1 week of treatment with 2 mg/kg siRNA. Stats: Welch's 1-way ANOVA * p<0.05, **** P<0.0001. Right: Serum Ttr assayed at time points indicated after 2mg/kg siRNA treatment (day 0). Liu et al., 2023 Nuc Acids Research

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Wave's new design for GalNAc-siRNA demonstrates improved durability in mice

	125											PBS
SerumTtr ±SEM (relto PBS)	100							*				Reference siRNA1
25										Liu et al. NAR 2023 design siRNA2
	75											New design siRNA
											*
	50									*
	0
	0	5	10	15	20	25	30	35	40	45	50
						Day
	0.5 mpk				Serum collection
	SubQ							7
1Foster, DJ. et.al. Mol Ther. 2018, 26(3), 708. 2 Liu W et al., Nucl Acids Res. 2023 51(9) C57BL/6 mice administered PBS or 0.5 mg/kg of siRNA (subcutaneous). Stats: Mixed
Two-way ANOVA followed by post hoc test comparing prior format siRNA vs. new format siRNA per day derived from linear mixed effects model * P < 0.0001

Wave's platform chemistry enables siRNA extrahepatic delivery

• Chemical impact of PN

- Introduction of neutral pKa backbone linkages

- Unique structural feature of PN, specifically guanidine, allows conjugation on oligonucleotide outside of 5' and 3' ends

- Increased lipophilicity

• Stereochemistry

• Extra-hepatic delivery

PN variants

- Titrating siRNA lipophilicity: tunable PNs (PN variants)

• Maintaining high Ago2 loading and intracellular trafficking
• Titrating plasma protein binding
• Altered delivery, enhanced potency and durability in various tissues

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Single dose of siRNA with PN variant 2 (PN-2) linkages delivers broad, potent and durable CNS target engagement in mice

D0	W8	W16
ICV 100 ug	Necropsy	Necropsy
App siRNA
Week 8				Week 16

Appsilencing

(SEM)remaining%mRNA (App/Hprt)

125

****

****

****

****

****

****

****

****

****

****

****

****

100

PBS

75

50

25

0

Cortex

Striatum

Cerebellum

Hippocampus

Brainstem

Spinal

cord

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PBS (dotted line) or 100 μg of App siRNA administered ICV (n=7). PCR assays for RNA PD, relative fold changes of App to Hprt mRNA normalized to % of PBS; Stats: Three-way ANOVA followed by Bonferroni-adjusted post hoc test comparing condition to PBS (data not shown), **** P < 0.0001 compared to PBS.

Single dose of siRNA incorporating PN-2 supports durable protein knockdown across the mouse CNS

Frontal Cortex

Striatum Hippocampus Cerebellum Brain Stem

PBS

App siRNA

PBS

App siRNA

D0W8W16

ICV 100 ug	Necropsy	Necropsy
App siRNA

8 weeks post-dose

16 weeks post-dose

Brown: App

Blue: Nuclei

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C57BL/6 mice were administered PBS or a single 100 μg intracerebral ventricular (ICV) injection of siRNA directed against mouse App. Mice were sacrificed at 8 and 16 weeks. Immunohistochemical analysis of fresh frozen paraffin-embedded mouse brain tissue labeling App protein (Brown). Nuclei were counterstained with Hematoxylin (Color Blue). Representative images are shown, magnification 100X.