RNA therapeutics in the treatment of retinal disease: delivering the potentialClinton Hall1, Adam Martin1, SingYee Yeung1, Hoang Vo1, Drew Innocend11, Mark Anastasas1, Tracy Chai1, Anja Stirnweiss1, Richard W Francis1, Suzy M Juraja1, Ferrer Ong1, Anna D Mills1, Laura Florez1, Maria Kerfoot1, Ben Wylie1,

Jessica Nicholls1, Danie Champain1, Paula T Cunningham1 and Sue Fletcher1,2

1PYC Therapeutics, Nedlands, WA, Australia.2Centre for Molecular Medicine and Innovative Therapeutics, Murdoch University, Murdoch, WA, Australia.

A major challenge to nucleic acid therapeutics is achieving efficient and targeted delivery

The opportunity:The highest value drug targets (DNA, RNA, protein) exist inside cells.

The challenge:The cell membrane has evolved over hundreds of millions of years to keep foreign substances out.

The Implication:Many breakthrough therapeutics fail due to an inability to reach the target.

Nucleic acid drugsare showing promise in a range of diseases but are hampered by inefficient delivery to target cells.

Antisense oligomer (AO) modulated gene expression

Limited scope for improvement

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Lead CPP-PMO Competitor CPP-PMONo retinal thinning Severe retinal thinning

* CPP-Cell penetrating peptide

High-performing cell penetrating peptides selected from our diverse peptide library deliver antisense oligomers (PMO) to the retina

We have developed proprietary screening methods and advanced filtering to identify CPPs with excellent efficacy and tolerability.

What are the libraries? Our libraries contain ~500 million unique peptides from 82 genomes and 118 synthetic viral genesDiverse : Derived from the genomes of bacteria, archaea and viruses, the peptide libraries are extremely diverseTargeted: The libraries have been enriched with viral genomes that have evolved to penetrate cells.

The libraries and machine learning models provide the flexibility to choose appropriate CPPs from our existing diverse panel and to further optimise for various cell and tissue types.

e) GFAP (marker of injury) expression in retina, 5 days after IVT injection of competitor and PYC CPP-PMOs. (f) OCT imaging 21 days after Smn CPP-PMO IVT injection in mice.

b) Efficient delivery of CPP-PMO to mouse retinal tissues in vivo, 7 days after IVT injection of 1.6 mg Smn CPP-PMO to induce Smn exon 7 skipping; with excellent uptake and duration of effect compared to unconjugated PMO (c). (d) CPP-PMO-induced Smn exon 7 skipping in patient iPSC derived retinal organoids.

Knockdown gene expression: transcript degradation -RNA silencing, RNaseH induced (DNA analgoues), disrupt the open reading frame (exon skipping)Alter isoforms: AO mediated exon/splice site selection (RNA analouges; 2’-O-modified phosphorothioate ‘ASO’, phosporodiamidate morpholino oligomers ‘PMO’) Block translation, enhance translation, alter mRNA stability: RNA analogues.

Approaches to targeted delivery of AOs

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Conventional approach to CPP developmentis to take previously poor-performing CPPs and attempt to increase potency or reduce cytotoxicity through incremental changes that limit diversity.

CPP-PMO delivery to retinal cells in vivo and to patient-derived cell models

a) Smn FL and E6/8 specific BaseScope TM staining of mouse eyes. Mouse was treated by IVT injection with the reporter Smn CPP-PMO that induces skipping of exon 7, 1.6 µg/eye, (28 days post treatment).

Image from Biorender.com

CPP-PMO delivery holds promise for the treatment of retinal disease

Disclosure: SF is a named inventor on intellectual property licensed to PYC Therapeutics.

Our discovery cell penetrating peptides are derived from nature, lack chemical modifications, and yield optimal amino acid sequences with enhanced efficacy and tolerability performance. Our lead CPP traffics the PMO through the vitreous, into the neural retina and retinal pigment epithelium, resulting in enhanced reporter exon skipping, with no evidence of retinal damage.

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Cold Spring Harb Perspect Biol. 2016 Jul; 8(7): a018069

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(d)Our peptide libraries are

derived from bacteria, archea, viruses

Animals, plants & fungi

Diverse Peptide Libraries

Screening in Mammalian Cells

High-Throughput Assays

Advanced Analytics and Machine Learning

in vivo Testing

Iterative Maturation

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Antisense oligomer mediated changes to gene expression

CPP-PMO evaluation in patient induced pluripotent stem cell-derived retinal organoids

Functional analysis

Modality Class ScalabilityTargeting capacity In vivo stability Immunogenicity

CPP Synthetic molecule Excellent Excellent Excellent LowAntibody Biological molecule Moderate Excellent Moderate HighLiposome Synthetic particle Moderate Poor Poor ModerateDendrimer Synthetic particle Excellent Poor Excellent ModerateNanoparticle Synthetic particle Excellent Moderate Excellent ModerateExosome Biological particle Poor Moderate Poor ModerateViral vector Biological particle Poor Poor Moderate High

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