Therapeutic RNA editing by Axiomer editing oligonucleotides

Transcription

1 Therapeutic RN editing by xiomer editing oligonucleotides

2 Forward looking statements This presentation contains forward-looking statements that involve substantial risks and uncertainties. ll statements, other than statements of historical facts, contained in this presentation, including but not limited to, statements regarding our strategy, future operations, future pre-clinical and clinical trial plans and related timing of trials and results, research and development, future financial position, future revenues, projected costs, prospects, therapeutic potential of our products, plans and objectives of management, are forward-looking statements. The words aim, anticipate, believe, estimate, expect, intend, may, plan, predict, project, target, potential, will, would, could, should, continue, and similar expressions are intended to identify forward-looking statements, although not all forward-looking statements contain these identifying words. Forward-looking statements represent our management s beliefs and assumptions only as of the date of this presentation. We may not actually achieve the plans, intentions or expectations disclosed in our forward-looking statements, and you should not place undue reliance on our forward-looking statements. ctual results or events could differ materially from the plans, intentions and expectations disclosed in the forward-looking statements we make. The forward-looking statements contained in this presentation reflect our current views with respect to future events, and we assume no obligation to update any forward-looking statements except as required by applicable law. These forward-looking statements are subject to a number of risks, uncertainties and assumptions, including those that may be described in greater detail in the annual report filed on Form 20-F for the year ended December 31, 2016 that we have filed with the U.S. Securities and Exchange Commission (the SEC ) and any subsequent filings we have made with the SEC. We have included important factors in the cautionary statements included in that annual report, particularly in the Risk Factors section, and subsequent filings with the SEC that we believe could cause actual results or events to differ materially from the forward-looking statements that we make. ProQR Therapeutics 2

3 Key points There is significant unmet need for genetic disorders caused by G-to- mutations The underlying mutations can be reversed through targeted -to-i editing by DR enzymes xiomer Editing Oligonucleotides (EONs) can direct site-specific editing by endogenous DRs Rational EON design relies both on computational and empirical approaches to achieve high potency and drug-like properties ProQR Therapeutics 3

4 Therapeutic potential ProQR Therapeutics 4

5 Unmet need for genetic diseases caused by G-to- point mutations Human disease-causing substitution mutations G-to- mutations >20,000 targets xiomer Most G-to- mutations require correction of the specific nucleotide for functional restoration -to-i results in a functional -to-g change, providing means for correcting G-to- mutations -to-i editing Original sequence restored ProQR Therapeutics 5

6 -to-i editing: Therapeutic opportunity The most prevalent editing event in human tissues denosine Inosine Guanosine dapted from Tan et al Nature 550: No sequence dependence 4 million -to-i sites in the human transcriptome, both in coding and non-coding RNs Extent of editing similar in most human tissues, making therapeutic editing feasible in all disease areas ProQR Therapeutics 6

7 DRs deaminate adenosine in dsrn DRs are active in both the nucleus and cytoplasm DR dsrbds DR Subtype Cellular location ctivity Expression RN DR deaminase domain DR1 p150 Cytoplasm Editing Interferoninducible DR1 p110 Nucleus Editing Constitutive DR2 Nucleus Editing Constitutive DR1 (p150 isoform) DR1 (p110 isoform) DR2 Nishikura 2016, Nat Rev Mol Cell Biol ProQR Therapeutics 7

8 xiomer is widely applicable Examples of different target RNs RN Effect on RN Effect of therapy (Pre-) mrn Protein coding ncrn rrn Correct nonsense mutations Correct missense mutations Correct splice sites Restore translation Restore protein Restore splicing of mrn Introns trn Modify codon lter protein function Regulatory sequences snrn lncrn mirn Modify protein binding Modify 2 RN structure lter regulatory sequence lter expression lter expression Modify target RN/DN/ protein binding Other ProQR Therapeutics 8

9 xiomer is widely applicable Stop codons as PoC for a wide class of disease indications RN Effect on RN Effect of therapy (Pre-) mrn Protein coding ncrn rrn Correct nonsense mutations Correct missense mutations Correct splice sites Restore translation Restore protein Restore splicing of mrn UG UG U (Stop) Introns Regulatory sequences trn snrn lncrn mirn Other Modify codon Modify protein binding Modify 2 RN structure lter regulatory sequence lter protein function lter expression lter expression Modify target RN/DN/ protein binding UIG UGI UII (Trp) Class of mutations resulting in complete loss of function First proof of concept for the xiomer approach ProQR Therapeutics 9

10 Targeted -to-i editing ProQR Therapeutics 10

11 DRs deaminate adenosine in dsrn Endogenous editing DR targets: denosines in dsrns with incomplete helices DR dsrbds GluR-B (R/G) GluR-B (Q/R) RN DR deaminase domain GluR-6 (R/G) 5HT 2C Fln (Q/R) Wahlstedt & Öhman 2011, WIREs RN ProQR Therapeutics 11

12 DRs deaminate adenosine in dsrn Endogenous editing DR targets: denosines in dsrns with incomplete helices DR dsrbds GluR-B (R/G) GluR-B (Q/R) RN DR deaminase domain GluR-6 (R/G) 5HT 2C Fln (Q/R) RN I Wahlstedt & Öhman 2011, WIREs RN How to recruit DR to therapeutically relevant sites that are not dsrn? ProQR Therapeutics 12

13 Targeted RN editing with DR xiomer EONs as a single-component therapeutic Natural editing Rosenthal/Zhang Stafforst xiomer Natural DR Fusion DR Guide RN Natural DR Guide RN RN EON Editing site Montiel-González et al. 2016, NR Cox et al. 2017, Science Wettengel et al. 2016, NR ProQR Therapeutics 13

14 EONs designed for targeted editing (1) Endogenous editing RN DR dsrbds RN DR deaminase domain I ProQR Therapeutics 14

15 EONs designed for targeted editing (2) EON forms double helix by Watson-Crick basepairing Endogenous editing EON-directed therapeutic editing RN RN EON DR dsrbds RN DR deaminase domain I ProQR Therapeutics 15

16 EONs designed for targeted editing (3) DR deaminates target in EON-target RN helix Endogenous editing EON-directed therapeutic editing RN RN EON DR dsrbds RN DR deaminase domain I RN EON I ProQR Therapeutics 16

17 EONs designed for targeted editing (4) dvantage over RN guides: Specificity Endogenous editing EON-directed therapeutic editing RN RN EON DR dsrbds RN I I DR deaminase domain RN EON I Editing site with RN guides is flexible Editing site with EONs is precise: No off-target editing even if deaminase domain shifts ProQR Therapeutics 17

18 EONs designed for targeted RN editing Functionality defined by sequence and chemistry Sequence defines target RN binding EON chemical modifications enable: Editing specificity Stability (nuclease resistance) Uptake into cells Provides Stability Enables Editing Total length: ~35 nt RN EON Backbone modifications enable DR binding, and disable off-target editing ProQR Therapeutics 18

19 xiomer EONs Correction of premature termination codons ProQR Therapeutics 19

20 EONs can restore ORFs In vitro proof of concept in a GFP reporter GFP W57X reporter in Hepa1-6 cells W57X Mutation Mutation treated with EON Up to 85% of transcript corrected by editing DR overexpression Transfection with 100 nm EON UG UIG Readout by Sanger sequencing of the RT- PCR product ProQR Therapeutics 20

21 Therapeutically relevant model: Hurler syndrome WT IDU gene IDU W402X mutant Hurler syndrome Mucopolysaccharidosis type I Lysosomes GGs Lysosomes GGs Mutations in the IDU gene Enzyme Deficiency of the lysosomal iduronidase enzyme RN RN ccumulation of glycosaminoglycans (GGs) IDU W402X mutation most common cause: UGG -> UG DN DN ProQR Therapeutics 21

22 Hurler mouse model for targeted editing Idua W392X mutant and EON Readouts for restored function Lysosomes GGs Enzyme RN EON DN RN sequence correction Enzymatic activity GG accumulation ProQR Therapeutics 22

23 EONs edit Idua mrn in vitro Idua W392X reporter construct in MEF cells with endogenous DR Sanger sequencing Non-treated EON 1 (100 nm) EON 2 (100 nm) ProQR Therapeutics 23

24 EONs restore iduronidase in vitro Idua W392X reporter construct in MEF cells with endogenous DR Proteins from EON-treated cells Incubation with fluorescent iduronidase substrate analog Fluorescent readout ProQR Therapeutics 24

25 EONs restore iduronidase in vitro Endogenous Idua in primary W392X mouse cells Effect in cells of different origin: Transfection of 100 nm EON into cells isolated from the W392X mouse Lung: Fibroblasts Liver: Fibroblasts Liver: Hepatocytes ProQR Therapeutics 25

26 EONs restore iduronidase in vivo Liver EON delivery in the W392X mouse EON in vivo delivery: N=2 5 mg/kg EON IV (Liposomes) 4 doses over 8 days Target: approximate GG level in WT mouse ProQR Therapeutics 26

27 EON design and optimization Combining computational and empirical approaches ProQR Therapeutics 27

28 EONs designed for targeted RN editing Functionality defined by sequence and chemistry Sequence defines target RN binding EON chemical modifications enable: Editing specificity Stability (nuclease resistance) Bioavailability Provides Stability EON Enables Editing RN Backbone modifications enable DR binding, and disable off-target editing ProQR Therapeutics 28

29 EONs designed for targeted RN editing Functionality defined by sequence and chemistry Sequence defines target RN binding EON chemical modifications enable: Editing specificity Stability (nuclease resistance) Bioavailability Provides Stability EON Enables Editing RN Backbone modifications enable DR binding, and disable off-target editing EONS RE MIXMERS OF 2 -OME, DN ND PS (ND UNDISCLOSED MODIFICTIONS) ProQR Therapeutics 29

30 EONs designed for targeted RN editing Functionality defined by sequence and chemistry Sequence defines target RN binding; EON chemical modifications enable: Editing specificity (2 -OMe vs. DN) Stability (nuclease resistance) Bioavailability Provides Stability EON Enables Editing RN Backbone modifications enable DR binding, and disable off-target editing ProQR Therapeutics 30

31 Structural basis for specificity Fit of nucleotide modifications into the catalytic site Editing disabling region 2 -OMe compatible with DR binding Editing enabling region 2 -OMe causes a steric clash with the active site RN and DN are compatible with catalysis Structural modelling provides a basis for further optimization of EONs DR2 (deaminase domain) Target adenosine Target RN EON Editing enabling region Editing disabling region(s) ProQR Therapeutics 31

32 EON specificity: Editing enabling region RN and DN are compatible with editing (2 -OMe is not) EON w/ RN Idua mrn EON w/ DN Idua mrn EON transfection (100 nm) with Idua W392X reporter construct in MEF cells. Neg. CTRL EON (Full 2 -OMe) Idua mrn ProQR Therapeutics 32

33 EON specificity: Editing disabling region 2 -OMe prevents editing of off-target adenosines 100 nm EON transfection with GFP W57X reporter and DR1 overexpression Readout by Sanger sequencing of the RT-PCR product ~85% editing at target ; no off-target editing ProQR Therapeutics 33

34 EONs designed for targeted RN editing Functionality defined by sequence and chemistry Sequence defines target RN binding; EON chemical modifications enable: Editing specificity Stability (2 -OMe, DN, PS) Bioavailability Provides Stability EON Enables Editing RN Backbone modifications enable DR binding, and disable off-target editing ProQR Therapeutics 34

35 EON stability: Editing enabling region Improved endonuclease resistance by replacing RN with DN EON w/ RN Idua mrn Time: EON w/ RN EON w/ DN PBS FBS PBS FBS CSF 2h 1d 2h 1d (days) EON integrity analysed by denaturing PGE after incubation in biological fluids in vitro EON w/ DN PBS: Control buffer * FBS: Fetal bovine serum Idua mrn * CSF: Human cerebrospinal fluid EONs additionally contain PS modifications, which improve exonuclease resistance ProQR Therapeutics 35

36 EONs designed for targeted RN editing Functionality defined by sequence and chemistry Sequence defines target RN binding; EON chemical modifications enable: Editing specificity Stability Bioavailability (PS and undisclosed) Provides Stability EON Enables Editing RN Backbone modifications enable DR binding, disable off-target editing, and enable bioavailability ProQR Therapeutics 36

37 EON bioavailability Improved in vivo delivery by optimizing PS content EON delivery study to WT mouse brain: 100 µg EON (naked) Intracerebroventricular (ICV) injections 48 h treatment Detection by ISH (Cy5- labeled probe specific to the EON) EON with 11 PS bonds EON with 19 PS bonds EON contains specific, undisclosed regions with PS bonds ProQR Therapeutics 37

38 EON bioavailability: Rational redesign Changing sugar chemistry to improve uptake Delivery with other oligo types indicates better uptake with another (undisclosed) sugar modification (Mod-X) E.g. naked oligo delivery (OT) to mouse lung: 2 -OMe Mod-X Can nucleotides with Mod-X be incorporated into EONs without compromising DR activity? ProQR Therapeutics 38

39 EON bioavailability: Rational redesign Modelling enables incorporation of improved sugar chemistry Enzymatic activity in vitro (Normalized to 2 -OMe EON) Computational modelling defines regions where Mod-X is tolerated Same Mod-X substitution level ProQR Therapeutics 39

40 Summary: EON-mediated mrn editing EON-directed editing of a specific by endogenous DR EON-directed editing restores function in vitro and in vivo EON optimization is based on computational and empirical approaches to achieve optimal combination of drug-like properties and DR engagement ProQR Therapeutics 40

41 The Team ProQR Therapeutics 41

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