Background Pathogenic mutations in the USH2A gene disrupt the production of usherin, a protein expressed in the photoreceptors where it is required for their maintenance (Liu et al., 2007).

Pathogenic mutations in the USH2A gene cause retinitis pigmentosa (RP). Exon 13 mutations are present in both non-syndromic and syndromic forms of RP. Exon 13 mutations are some of the most common USH2A mutations and are estimated to be present in around 16,000 patients in the Western World (US, Canada, Europe and Australia). Currently there is no treatment available for RP caused by mutations in exon 13 of the USH2A gene.

QR‑421a is a 21‑mer single‑stranded chemically modified antisense oligonucleotide (AON). QR‑421a is designed to bind to a specific sequence in the USH2A pre-mRNA and modulate splicing by enhancing exon-skipping. Skipping of exon 13 from the USH2A mRNA results in an in-frame transcript that is expected to be translated into functional (albeit shorter) usherin protein.

It is hypothesized that treatment with QR-421a will result in restoration of functional usherin protein in photoreceptors and restoration of vision in patients with RP due to mutations in exon 13 of the USH2A gene.QR-421a for RP in Usher syndrome

ProQR Therapeutics 1

Skipping of exon 13 in USH2A mRNA

In wild type cells usherin enables protein transport through the

connecting cilium

In cells with the USH2A mutation usherin is not active, hampering protein transport over the cilium

Exclusion of the exon harboring the mutation leads to restoration of

functionality of usherin

Outersegment

Innersegment

Nucleus

Connectingcilium

pre-mRNA

DNA

QR-421amRNA

Exon 12 Exon 13 Exon 14

Exon 12 Exon 13 Exon 14

Exon 14Exon 12 Exon 13

Exon 12 Exon 14Exon 13

Exon 14Exon 12

Exon 12 Exon 14Exon 13

mRNA

pre-mRNA

QR-421a

Mutations in exon 13 of the USH2A gene result in the absence of the usherin protein in the retinal photoreceptors and degeneration of the outer segment of photoreceptor cells. QR-421a (in yellow) hybridizes to the USH2A pre-mRNA and results in exclusion of exon 13 from the mRNA, which leads to the production of a functional (shorter) usherin protein. It is hypothesized that restoration of usherin protein prevents degeneration of the outer segment of the photoreceptor.

Objectives• Assess in vitro and in vivo efficacy of QR-421a in retinal organoids

and cynomolgus monkeys by quantification of USH2A exon skip.

• Confirm functionality of usherin protein following skip of exon 13 using AONs in zebrafish.

Materials and Methods Retinal organoids are a clinically relevant system to thoroughly interrogate the mechanisms of inherited retinal degeneration. Fibroblasts from a healthy donor and a patient homozygous for USH2A c.2299delG (exon 13) mutation were first reprogrammed into iPSCs, which were then differentiated into retinal organoids for 60 days and then treated with QR-421a. Untreated organoids and organoids treated with a control oligonucleotide (with the same chemistry and length but a random sequence) were used as negative controls.

In the retina of cynomolgus monkeys QR-421a mediated exon 12 (equivalent to human exon 13) skip was assessed. Wild-type cynomolgus monkeys received single IVT injections (bilateral) of low, mid and high doses of QR-421a. Retina samples were collected at multiple time points post-injection (1 hour, 12 hours, 15 days, 28 days and 102 days) for assessment of exon skip. Retinas were separated from the eyes, RNA was isolated and the levels of USH2A transcripts with and without exon 12 were quantified using isoform specific ddPCR assays and the percentage of exon skip was calculated.

A zebrafish model, homozygous for exon 13 premature stop codon mutation (referred as to ush2armc1), was developed to assess the activity of the usherin protein resulting from the exon 13 skip in mRNA. Ush2armc1 zebrafish larvae, as expected, have no functional usherin protein and show a significantly reduced b-wave amplitude in electroretinogram (ERG) recordings (Dona et al, 2018). Ush2armc1 zebrafish were treated with zebrafish‑specific oligonucleotides followed by assessment of exon skip, usherin protein localization and recording of the ERG b-wave amplitude. Usherin protein was localized in radial cryosections of zebrafish larval (5 dpf) retinae using anti‑Ush2a antibodies directed against the intracellular C‑terminal tail of zebrafish usherin (red signal). Nuclei were stained with DAPI (blue signal).

Wild-type mice received bilateral IVT injection of QR-421a to assess in vivo delivery, eyes were fixed overnight in Hartmann’s fixative and embedded in paraffin.QR-421a was visualized in retina using complementary probe with Cy5 label by in situ hybridization. Images were acquired on a LSM800 confocal microscope.

Results Assay CharacteristicsConcentration-dependent Increase of USH2A Exon 13 Skip After one Month of Exposure to QR-421a in Patient Retinal Organoids

Untreated

Day 1 Day 28

10 µM Control

1 µM QR-421a

2 µM QR-421a

5 µM QR-421a

10 µM QR-421a

0

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40

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Patient retinal organoids

Perc

enta

ge∆e

xon

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)

QR-421a concentration

To determine the ability of QR-421a to induce exon 13 skip in USH2A, patient retinal organoids were treated continuously with QR-421a for 28 days. Untreated organoids and organoids treated with a control oligonucleotide (with the same chemistry and length but a random sequence) were used as negative controls.

USH2A transcripts with or without exon 13 were quantified by ddPCR and percentage of exon 13 exclusion was calculated. Results showed that QR-421a induced levels of exon 13 skip at all concentrations tested. At 1 µM concentration, 42% of exon 13 skip was observed which increased to 63 % at 10 µM concentration. No exon 13 skip was measured in untreated or control treated retinal organoids. Data shown as mean ± SEM.

Results (continued)

QR-421a, an antisense oligonucleotide, for the treatment of retinitis pigmentosa due to USH2A exon 13 mutationsHester van Diepen1, Kalyan Dulla1, Heelam Chan1, Iris Schulkens1, Wouter Beumer1, Lars Vorthoren1, Cathaline den Besten1, Levi Buil1, Iris Schmidt1, Janne Turunen1, Jiayi Miao1, Erik de Vrieze2, Sanne broekman2, Margo Dona2, Silvia Albert3, Erwin van Wijk2, Peter Adamson1

1 ProQR Therapeutics, Leiden, the Netherlands | 2 Otorhinolaryngology, Radboudumc, Nijmegen, Netherlands | 3 Human Genetics, Radboudumc, Nijmegen, Netherlands

USH2A Exon 13 Skip After QR-421a Single Dose Treatment Regimen Mimicking in vivo Situation in Wild-type Retinal Organoids

Untreated 10 µM Control

1 µM QR-421a

5 µM QR-421a

10 µM QR-421a

0

20

40

60

80

Perc

enta

ge∆e

xon

13(%

)Day 1 Day 28

QR-421a

ns

ns*

As a follow-up experiment, the treatment conditions mimicked vitreous concentrations following administration of a single IVT injection. Retinal organoids were treated with (1, 5 or 10 µM) QR‑421a, after which half of the medium was replenished every two days with medium without AON, to gradually decrease the QR-421a concentration in the organoid culture. With this serial dilution QR-421a is completely removed from the medium by day 12. This change in concentration in the culture medium mimics the kinetics in vitreous humor. USH2A transcripts with or without exon 13 were quantified by ddPCR and percentage of exon 13 exclusion was calculated. Data is shown as mean ± SEM. QR-421a treatment resulted in a dose-dependent exon skip in retinal organoids, and interestingly a significant amount of exon 13 skip was noticed even 2 weeks after removing all QR-421a from the medium.

Dose-dependent USH2A Exon Skip by QR-421a in Wild-type Cynomolgus Monkey Retina

PBS low mid high0

20

40

60

80

USH2A ∆exon 12 (%)

Dose of QR-421a

USH

2A∆e

xon

12(%

)

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USH2A ∆exon 12 (%)

Low Mid High

USH

2A∆e

xon

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)

Dose of QR-421a

28 days post IVT

A B

Pharmacodynamic activity of QR‑421a was confirmed in vivo. Cynomolgus monkeys received a single bilateral intravitreal injection as part of toxicology studies and were maintained for 28 days. USH2A RNA analysis was done by isoform specific ddPCR. Summary data is shown in the left graph and individual values are shown in the right graph. Data is shown as mean ± SEM. QR-421a induced dose-dependent USH2A skip. At the low, mid and high doses 26 %, 48 % or 69 % USH2A skip was detected respectively 28 days post IVT injection. No exon skip (<0.5 %) was detected in PBS treated animals.

Duration of Action of QR-421a in Wild-type Cynomolgus Monkey Retina Maintained for at least 102 days

1 hour 12 hours 15 days 102 days0

5

10

15

20

USH2A ∆exon 12 (%)

Days post IVT

USH

2A∆e

xon

12(%

)

0

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USH2A ∆exon 12 (%)

Days post IVT

USH

2A∆e

xon

12(%

)

15 days 102 days

Dose 1 QR-421aDose 2 QR-421a

A B

Cynomolgus monkeys received a single bilateral intravitreal injection of QR-421a and were maintained up to 102 days post IVT injection. USH2A exon skip was determined 1 hour, 12 hours, 15 days and 102 days post injection. Summary data is shown in the left graph and individual values are shown in the right graph. Data is shown as mean ± SEM. QR-421a induced dose-dependent exon skip at all time-points, which increased over time. This data provides in vivo evidence for QR-421a mediated dose-dependent USH2A exon skip, maintained for at least 102 days post single IVT injection.

Restoration of Usherin Protein Localization and ERG b-Wave in a Mutant Zebrafish Model After AON Mediated ush2a Exon 13 Skip

G C A A G G C A A A C G T A A T C G G T T T T T T C C T GT C C G A G G

1096 bp

ush2a Δex13

ush2a WT

448 bp

uninjected

WT (3 dpf)ush2armc1 (3 dpf)

ush2a exon 12 ush2a exon 14

uninjected mQMO-N1 +MO-P2

MO-N1 +MO-P3

This ush2armc1 zebrafish model was used to assess the potential to restore usherin protein expression and retinal function following AON mediated exon 13 exclusion. Ush2a RT-PCR analysis showed that use of a combination of two oligonucleotides (MO‑N1 + MO‑P2 or MO‑N1 + MO‑P3) that target different regions of ush2a exon 13, resulted in a near complete skip of ush2a exon 13. Sanger sequencing of the resulting PCR amplicon after oligonucleotide injections revealed a perfect skipping of exon 13.

IPL

WT Ush2armc1 + MO

OPS

ONL

IS

OSCC region

Ush2armc1

The oligonucleotide combinations were then applied to homozygous ush2armc1 larvae to determine whether this exon 13 skip had an effect on the phenotypic outcome of the ush2armc1 mutant. In wild-type larvae usherin is present at the base of the photoreceptor connecting cilium. In homozygous ush2armc1 larvae, no specific usherin signal could be detected. Oligonucleotide‑induced exon 13 skipping in homozygous ush2armc1 larvae resulted in a partial restoration of usherin expression and localization in photoreceptor cells of ush2armc1 larvae.

-300

-200

-100

0

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A

b w

ave

ampl

itude

(µV)

time (ms)B

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ush2a rmc1wild-type ush2a rmc1 ush2a rmc1 ush2a rmc1

+ control MO + MO-N1+P2 + MO-N1+P3

**

**

Max

b w

ave

ampl

itude

(µV)

1 28 55 82 109

136

163

190

217

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271

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379

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541

568

595

622

649

676

703

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784

811

838

865

892

919

946

973

1000

wild-type (n = 10)ush2a (n = 11)rmc1

ush2a + control MO (n = 14)rmc1

ush2a + MO-N1 + P2 (n = 14)rmc1

ush2a + MO-N1 + P3 (n = 25)rmc1

To measure retinal function, ERG recordings were performed in ush2armc1 larvae in which ush2a exon 13 skipping was induced by oligonucleotide treatment. Top graphs show average ERG b-wave traces from uninjected, control MO injected, exon 13 skipping MO injected homozygous ush2armc1 larvae (5 dpf) with wild-type controls. Bottom panel shows mean of the maximum ERG b-wave amplitudes are plotted as bar graphs with standard deviation. ERG recordings in ush2armc1 larvae in which exon skipping was induced showed a significantly improved b-wave amplitude compared to untreated larvae and larvae treated with control oligonucleotides.

In vivo Uptake and Retention of QR-421a in the Outer Nuclear Layer of the Retina

4 weeks

1 hour

8 weeks

48 hours

PBS 48 hours

2 weeksONL

ONLONL

ONL

ONL

INL

GCL 20 μm

RPE

ONL

OSIS

INL

GCL

Retinal cell layers

48 hours

ONL

INL

GCL

ONL

To confirm the delivery, we have visualized QR‑421a in the retinal outer nuclear layer (ONL), which comprises of photoreceptor nuclei, using in situ hybridization. Wild‑type C57Bl/6 mice received a single bilateral 1 μl intravitreal injection of 30 μg of QR‑421a and were maintained for 2, 14, 28, or 56 days. Clear QR‑421a signal was detected in the ONL at all time points tested here confirming delivery and indicating long half-life. No signal was detected in the PBS treated retina.

Conclusions• QR-421a induces dose-dependent USH2A exon skip in vitro in

retinal organoids and in vivo in cynomolgus monkeys maintained for at least 3 months post single intravitreal injection in cynomolgus monkeys.

• Exon 13 skipping at the mRNA level was shown to result in restoration of usherin protein expression and restoration of ERG in an ush2a mutant zebrafish model.

References1. Dona M, Slijkerman R, Lerner K, Broekman S, Wegner J, Howat T et al. Usherin

defects lead to early‑onset retinal dysfunction in zebrafish. Experimental eye research. 2018; 173:148-159.

2. Liu X, Bulgakov OV, Darrow KN, Pawlyk B, Adamian M, Liberman MC, et al. Usherin is required for maintenance of retinal photoreceptors and normal development of cochlear hair cells. Proc Natl Acad Sci USA 2007; 104(11):4413 8.

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Wild-type RP RP + QR-421a