rna therapeutics in the treatment of retinal disease
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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
G R K K R R Q R R R P Q
- R K K R R Q R R R P Q
G R R K R R Q R R R P Q
Y R K K R R Q R R R P Q
Y R R R R R Q R R R P Q
TAT
TAT1
TAT2
TAT3
TAT4
TAT TAT1 TAT2 TAT3 TAT40
20
40
60
80
100 Cell Entry
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
*
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.
Smn CPP-PMO treatedUntreated
30
20
10
Ganglion
cell layer
Rods &
cones
Image created on Biorender.com
0 10 20 30
TAT
1st Gen
2nd Gen
Matured
Fold TAT
Cargo Delivery to ARPE-19 cells
Cold Spring Harb Perspect Biol. 2016 Jul; 8(7): a018069
(b) (c)
(a)
Untreated 5 mM 10 mM CPP-PMO n=4 n=4 n=1
Exon
skip
ping
(%)
80
60
40
20
(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
(e) (f)
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
Untreated CPP-PMO treated
Phag
ocyt
osis:
Mea
n flu
ores
cenc
e in
tens
ity
Ganglioncell layer
Inner plexi-form layer
Inner nuclear layer
Outer nuclear layer
PYC CPP 1 PYC CPP 2 PMO only0
20
40
60
80
100
56
35
0
59
35
0
Exon Skipping in the Retina1.6 ug/eye, Day 7 vs Day 28 Timepoint
Trun
cate
dSm
n tr
ansc
ript (
%) 7-day
28-day
PYC CPP 1 PYC CPP 2 PMO only0
20
40
60
80
100
2919
0
59
35
0
Exon SkippingDay 7, 1.6 ug/eye
Trun
cate
dSm
n tr
ansc
ript (
%) Neural Retina
RPE layer
Exon
skip
ping
(%)
100
80
60
40
20
CPP-PMO1 CPP-PMO2 PMO only
Exon
skip
ping
(%)
CPP-PMO1 CPP-PMO2 PMO only
100
80
60
40
20
Competitor
PYC PPMO1
PYC PPMO2
PYC PPMO30.0
0.1
0.2
0.3 GFAP Score - 5 days
Competitor CPP CPP CPP CPP-PMO PMO1 PMO2 PMO3
0.2
0.1
Untreated PYC PPMO
Phag
ocyt
osis
: Med
ian
Fluo
resc
ence
Inte
nsity
p<0.0001
TAT TAT1 TAT2 TAT3 TAT40
20
40
60
80
100 Cell Entry80
60
40
20
TAT TAT1 TAT2 TAT3 TAT4
Exp29
_001
Exp29
_002
Exp29
_003
Exp29
_004
Exp29
_005
Exp29
_006
Exp29
_007
Exp29
_008
Exp29
_009
full-d-R
8
full-d-R
10
PYC contro
l
Competitor
PMO only
No treatm
ent
0
20
40
60
80
100
d7 (%
)
SMN1 PPMOExon Skipping in ARPE-19 Cells
1uM2uM4uM
CPP -01 -02 -03 -04 -05 -06 -07 -08 -09 full-d- full-d- PYC competitor PMO untreated R8 R10 control only
100
80
60
40
20
SMN1 exon 7 skipping PPMO-treated ARPE-19 cells
Matured
2nd Gen
1st Gen
TAT
0 10 20 30Fold TAT
Ganglioncell layer
Rods &cones
0
10
20
30
UntreatedSmn PPMO
Exon
skip
ping
(%)
GFAP
Exon
skip
ping
(%)
Cell
entry
(%
)
Cargo delivery to ARPE-19 cells
Rods & cones
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