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8-K

 

UNITED STATES

SECURITIES AND EXCHANGE COMMISSION

Washington, D.C. 20549

 

 

FORM 8-K

 

 

CURRENT REPORT

Pursuant to Section 13 or 15(d)

of the Securities Exchange Act of 1934

Date of Report (Date of earliest event reported): May 12, 2020

 

 

Stoke Therapeutics, Inc.

(Exact Name of Registrant as Specified in its Charter)

 

Delaware   001-38938   47-114582

(State or other jurisdiction of

incorporation or organization)

  

(Commission

File Number)

  

(I.R.S. Employer

Identification No.)

45 Wiggins Ave

Bedford, Massachusetts

     01730
(Address of principal executive offices)     (Zip Code)

Registrant’s telephone number, including area code: (781) 430-8200

Not Applicable

(Former Name or Former Address, if Changed Since Last Report)

Check the appropriate box below if the Form 8-K filing is intended to simultaneously satisfy the filing obligation of the registrant under any of the following provisions:

 

☐

Written communications pursuant to Rule 425 under the Securities Act (17 CFR 230.425)

 

☐

Soliciting material pursuant to Rule 14a-12 under the Exchange Act (17 CFR 240.14a-12)

 

☐

Pre-commencement communications pursuant to Rule 14d-2(b) under the Exchange Act (17 CFR 240.14d-2(b))

 

☐

Pre-commencement communications pursuant to Rule 13e-4(c) under the Exchange Act (17 CFR 240.13e-4(c))

Securities registered pursuant to Section 12(b) of the Act:

 

Title of each class

  

Trading Symbol(s)

  

Name of each exchange on which registered

Common Stock, $0.0001 par value per share   STOK   Nasdaq Global Select Market

 

 

 

Indicate by check mark whether the registrant is an emerging growth company as defined in Rule 405 of the Securities Act of 1933 (§230.405 of this chapter) or Rule 12b-2 of the Securities Exchange Act of 1934 (§240.12b-2 of this chapter).

Emerging growth company ☒

If an emerging growth company, indicate by check mark if the registrant has elected not to use the extended transition period for complying with any new or revised financial accounting standards provided pursuant to Section 13(a) of the Exchange Act. ☐

Item 7.01

Regulation FD

In connection with the 2020 Annual Meeting of the American Society of Gene & Cell Therapy (“ASGCT”), on May 12, 2020, Stoke Therapeutics, Inc. (the “Company”) issued the press release attached as Exhibit 99.1. In addition, on May 12, 2020, the poster attached as Exhibit 99.2, which the Company will present at the ASGCT Annual Meeting, was posted to the ASGCT website.

The information furnished with this report, including Exhibit 99.1, shall not be deemed “filed” for purposes of Section 18 of the Securities Exchange Act of 1934, as amended (the “Exchange Act”), or otherwise subject to the liabilities of that section, nor shall it be deemed incorporated by reference into any other filing under the Exchange Act or the Securities Act of 1933, as amended, except as expressly set forth by specific reference in such a filing.

 

Item 9.01

Financial Statements and Exhibits.

(d) Exhibits

 

Exhibit

Number

   

Description

99.1    Press Release, dated May 12, 2020.
99.2    ASGCT Poster.

SIGNATURE

Pursuant to the requirements of the Securities Exchange Act of 1934, the registrant has duly caused this report to be signed on its behalf by the undersigned hereunto duly authorized.

 

    STOKE THERAPEUTICS, INC.
Date: May 12, 2020     By:   /s/ Robin A. Walker
     

Robin A. Walker

Senior Vice President, Chief Legal Officer and Chief Compliance Officer

EX-99.1

Exhibit 99.1

Stoke Therapeutics Presents Preclinical Data That Demonstrate In-Vitro and In-Vivo Target Engagement and Protein Upregulation in OPA1 Protein Deficiency, the Underlying Cause of the Most Common Inherited Optic Nerve Disorder

First in-vivo proof-of-concept for TANGO antisense oligonucleotides in an ocular disease

Results presented at the American Society of Gene and Cell Therapy Annual Meeting further validate the company’s mutation-independent approach to amplifying protein expression to treat severe genetic diseases

BEDFORD, Mass., May 12, 2020 — Stoke Therapeutics, Inc., (Nasdaq: STOK), a biotechnology company pioneering a new way to treat the underlying cause of genetic diseases by precisely upregulating protein expression, today announced new preclinical data demonstrating in-vitro and in-vivo target engagement and protein upregulation in OPA1 protein-deficient cells. OPA1 protein deficiency is the underlying cause of autosomal dominant optic atrophy (ADOA), the most common inherited optic nerve disorder. This is the first proof-of-concept data for TANGO antisense oligonucleotides (ASOs) in an ocular disease. The results further validate the company’s mutation-independent approach to amplifying protein expression to treat severe genetic diseases. These data will be presented today in a virtual poster session at the American Society of Gene and Cell Therapy (ASGCT) 2020 Annual Meeting.

“These data provide early evidence of the potential to address the underlying cause of autosomal dominant optic atrophy, an optic nerve disorder that causes progressive and irreversible vision loss starting in the first decade of a child’s life. There are currently no approved treatments for ADOA,” said Edward M. Kaye, M.D., Chief Executive Officer of Stoke Therapeutics. “Our TANGO technology represents a unique, mutation-independent approach to treating the underlying cause of a variety of genetic diseases, particularly in the central nervous system and the eye. The ADOA program is one of several under consideration for future prioritization, and we look forward to nominating a second product candidate later this year.”

ADOA affects approximately one in 30,000 people globally with a higher incidence in Denmark of one in 10,000 due to a founder effect. An estimated 65% to 90% of cases are caused by mutations in the OPA1 gene.

The data presented today demonstrate in-vitro and in-vivo proof-of-concept for TANGO ASOs in an ocular disease. Highlights from today’s presentation include:

 

  •  

Dose-dependent decreases in non-productive OPA1 mRNA and increases in OPA1 protein expression were observed in-vitro and in-vivo.

 

  •  

An increase in OPA1 protein expression to approximately 75% of wild-type levels was observed in an OPA1 haploinsufficient (OPA1 +/-) cell line.

 

  •  

In-vivo increases in OPA1 protein levels in the retina of wild-type rabbits were observed and the test ASO was well tolerated for up to 15 days after intravitreal injection.

Details of today’s presentation are as follows:

Presentation Title: Antisense oligonucleotide mediated increase of OPA1 expression using TANGO technology for treatment of autosomal dominant optic atrophy

Session Date & Time: Tuesday, May 12, 2020; 5:30 p.m. – 6:30 p.m. E.T.

Session Title: Oligonucleotide Therapeutics

Presenter: Aditya Venkatesh, Ph.D., Senior Scientist, Stoke Therapeutics

The poster presented at ASGCT is now available online on the Events and Presentations section of Stoke’s website at https://investor.stoketherapeutics.com/.

About Autosomal Dominant Optic Atrophy

Autosomal dominant optic atrophy (ADOA) is the most common inherited optic nerve disorder. It is a rare disease that causes progressive and irreversible vision loss in both eyes starting in the first decade of life. Symptoms typically begin between the ages of 4 and 6 years old, affecting males and females equally. The severity of the condition by adolescence reflects the overall level of visual function to be expected throughout most of the individual’s adult life. Roughly half of people with ADOA fail driving standards and up to 46% are registered as legally blind. ADOA is considered a haploinsufficiency, as most people living with ADOA have genetic mutations in the OPA1 gene that result in only half the necessary OPA1 protein being produced. More than 400 OPA1 mutations have been reported in people diagnosed with ADOA. Currently there is no approved treatment for people living with ADOA.

About TANGO

TANGO (Targeted Augmentation of Nuclear Gene Output) is Stoke’s proprietary research platform. Stoke’s initial application for this technology are diseases in which one copy of a gene functions normally and the other is mutated, also called haploinsufficiencies. In these cases, the mutated gene does not produce its share of protein, so the body does not function normally. Using the TANGO approach and a deep understanding of RNA science, Stoke researchers design antisense oligonucleotides (ASOs) that bind to pre-mRNA and help the target genes produce more protein. TANGO aims to restore missing proteins by increasing – or stoking – protein output from healthy genes, thus compensating for the non-functioning copy of the gene.

About Stoke Therapeutics

Stoke Therapeutics (Nasdaq: STOK), is a biotechnology company pioneering a new way to treat the underlying causes of severe genetic diseases by precisely upregulating protein expression to restore target proteins to near normal levels. Stoke aims to develop the first precision medicine platform to target the underlying cause of a broad spectrum of genetic diseases caused by haploinsufficiencies. Stoke is headquartered in Bedford, Massachusetts with offices in Cambridge, Massachusetts. For more information, visit https://www.stoketherapeutics.com/ or follow the company on Twitter at @StokeTx.

Cautionary Note Regarding Forward-Looking Statements

This press release contains “forward-looking” statements within the meaning of the “safe harbor” provisions of the Private Securities Litigation Reform Act of 1995, including, but not limited to: Stoke’s ability to precisely upregulate protein expression in OPA1 protein-deficient cells; Stoke’s ability to treat the underlying cause of ADOA; and Stoke’s ability to use preclinical data to advance the development of TANGO ASOs to treat ocular disease. Statements including words such as “plan,” “continue,” “expect,” “target,” or “ongoing” and statements in the future tense are forward-looking statements. These

forward-looking statements involve risks and uncertainties, as well as assumptions, which, if they do not fully materialize or prove incorrect, could cause our results to differ materially from those expressed or implied by such forward-looking statements. Forward-looking statements are subject to risks and uncertainties that may cause Stoke’s actual activities or results to differ significantly from those expressed in any forward-looking statement, including without limitation risks and uncertainties related to Stoke’s ability to develop, obtain regulatory approval for and commercialize TANGO ASOs to treat ocular disease; the impact of the COVID-19 pandemic on Stoke’s operations and the U.S. and world economies; the timing and results of preclinical studies; the timing for nominating a second product candidate; risks associated with potential delays, work stoppages, or supply chain disruptions caused by the coronavirus pandemic; risks associated with current and potential future healthcare reforms; Stoke’s ability to protect its intellectual property; and other risks set forth in our filings with the Securities and Exchange Commission, including the risks set forth in our quarterly report on Form 10-Q for the quarter ended March 31, 2020. These forward-looking statements are based on our current beliefs and expectations and speak only as of the date hereof and Stoke specifically disclaims any obligation to update these forward-looking statements or reasons why actual results might differ, whether as a result of new information, future events or otherwise, except as required by law.

Stoke Media & Investor Contact:

Dawn Kalmar

Vice President, Head of Corporate Affairs

dkalmar@stoketherapeutics.com

781-303-8302

EX-99.2

Exhibit 99.2 Antisense oligonucleotide mediated increase of OPA1 expression using TANGO technology for treatment of autosomal dominant optic atrophy 1 2 1 1 1 1 2 1 1 1 Aditya Venkatesh , Zhiyu Li , Syed Ali , Anne Christiansen , Kian Huat Lim , Jacob Kach , Robert B. Hufnagel , Jeffrey H. Hoger , Isabel Aznarez , Gene Liau 1 2 Stoke Therapeutics, Bedford, MA, Medical Genetics and Ophthalmic Genomics Unit, National Eye Institute, National Institutes of Health, Bethesda, MD Rabbit surrogate ASO decreases non-productive splicing and increases OPA1 Specific ASOs reduce non-productive splicing Background expression in wild-type rabbit retinae following intravitreal injection and increase productive OPA1 mRNA levels in vitro Autosomal dominant optic atrophy (ADOA) is one of the most commonly Final conc. Dose A. Study Design A. Dose Dose and Matrices RT-PCR for non-productive OPA1 mRNA Group N Treatment expected in volume diagnosed optic neuropathies. This optic nerve disease is associated with (ug/eye) regimen collected (OU) vitreous* (uM) (uL/eye) Day 1 No ASO 1 3 Vehicle (PBS) NA N/A structural and functional mitochondrial deficits that lead to degeneration Retinae split Single intravitreal injection of along the 2 3 NT ASO 110 12 vehicle/ASO (n=3/group) IVT OU nasal-temporal of the retinal ganglion cells and progressive, irreversible loss of vision. A Test ASO – 30 on Day 1 axes, temporal 3 3 39 4 Female NZW rabbits A X B Low Dose half used for majority of ADOA patients carry mutations in OPA1 and most mutations RNA, nasal for Test ASO – Day 16 4 3 116 12 protein High Dose A B Euthanize animals and collect retinal tissue lead to haploinsufficiency (Lenaers G. et al. Orphanet J Rare Dis 2012). B. Non-productive OPA1 mRNA C. OPA1 encodes a mitochondrial GTPase with a critical role in mitochondrial B. Non-productive OPA1 mRNA Quantification C. Productive OPA1 mRNA – Taqman qPCR Test ASO Test ASO NT ASO Non-productive OPA1 mRNA OPA1 protein Vehicle (PBS) 4uM 12uM 12uM 1.4 * fusion, ATP synthesis and apoptosis. Currently, there is no approved 16 25 1.4 U499 U514 U516 U501 U505 U524 U517 U518 U520 U503 U513 U521 1.2 14 OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS 1.2 20 1.0 disease-modifying treatment for ADOA patients. Here, we employ TANGO 12 1.0 15 0.8 10 A X B 0.8 (Targeted Augmentation of Nuclear Gene Output), a novel therapeutic 0.6 8 10 * 0.6 0.4 6 0.4 A B 5 approach, that uses antisense oligonucleotides (ASOs), to increase the 4 0.2 * 0.2 2 0.0 0 endogenous expression of OPA1. 0.0 0 OPA1 protein Figure 1: Mechanism of TANGO: OPA1 TANGO reduces non-productive Figure 4. For in vitro screening, ASOs were transfected at 80nM dose into HEK293 cells using Lipofectamine RNAiMax as messenger RNAs (mRNA), which are a transfection agent. For effect on NMD exon, cells were treated with CHX (50ug/mL, 3 hrs.) 21 hours after transfection. β-actin normally targeted for degradation by RNA was isolated and used for RT-PCR (Panel A with quantification in Panel B). For OPA1 mRNA expression, non- nonsense-mediated mRNA decay (NMD) cycloheximide treated cells were used for Taqman qPCR and mRNA expression of OPA1 was normalized to RPL32. Red Figure 7. Rabbits were used as a surrogate for initial in vivo proof of concept studies to test if our ASO can increase OPA1 as shown in Figure 1. In turn, TANGO arrows highlight ASOs that reduce non-productive splicing and increase OPA1 mRNA expression by at least 20%. expression in the retina following intravitreal injection. Female New Zealand White (NZW) adult rabbits were injected increases productive mRNA and protein. Among these, ASO-14 produces the most increase in OPA1 mRNA (30%) with either vehicle, non-targeting (NT) or test ASO, and animals were euthanized after 15 days to obtain retinal tissue. TANGO specifically increases expression Panel A outlines the study design while Panels B and C depict impact on NMD exon and OPA1 expression (* P<0.05 by ASO-14 decreases non-productive OPA1 mRNA and increases OPA1 of canonical target mRNA and full-length one-way ANOVA compared to Vehicle group). Data show that following intravitreal injection in the rabbit eye, our test protein, only in tissues with endogenous ASO reduces non-productive OPA1 mRNA and increases OPA1 expression in retinal tissue. expression in a dose-dependent manner in vitro gene expression. As these events are OD: oculus dextrus (right eye); OS: oculus sinister (left eye); OU: oculus uterque (both eyes) A. Non-productive OPA1 mRNA B. Productive OPA1 mRNA C. OPA1 protein naturally-occurring, TANGO can *Final concentration in the vitreous calculated assuming vitreal volume in the rabbit as 1.5mL * 1.4 1.4 25 upregulate the wild-type alleles in the * * 1.2 1.2 context of autosomal dominant 20 * Conclusions 1.0 1.0 haploinsufficiency diseases such as 15 0.8 0.8 ADOA. 0.6 0.6 We have validated TANGO as a novel therapeutic approach to address 10 * 0.4 0.4 ADOA caused by OPA1 haploinsufficiency 5 * 0.2 0.2 0.0 0.0 0 Additionally, TANGO offers the following advantages for treating ocular diseases: ✓ Intravitreal injection of ASOs permits diffusion throughout the eye, including retinal neurons ✓We identified ASOs that reduce non-productive OPA1 splicing, increase NT ASO ASO-14 NT ASO ASO-14 NT ASO ASO-14 ✓ Long-term efficacy (>1 year in mouse retina) after single intravitreal injection (Kach et al, productive OPA1 mRNA, and increase OPA1 protein levels ARVO Poster Presentation May 2019) A X B OPA1 +/- ✓ASO-14 increased OPA1 protein up to ~75% of wild-type levels in OPA1 ✓ No specialized formulation or encapsulation required for ASO therapy β-actin ✓ Potential to target large genes not amenable to AAV-based gene therapy HEK293 cells A B ✓The rabbit surrogate ASO increased OPA1 protein in vivo in wild-type Figure 5. HEK293 cells were transfected with different doses of ASO-14 or non-targeting (NT) ASO. RNA was isolated 24 OPA1 non-productive splicing event identification and validation hours after transfection and analyzed for impact on non-productive OPA1 mRNA (Panel A) and OPA1 mRNA expression rabbit retinae and was well tolerated for up to 15 days after intravitreal (Panel B) similar to Figure 4. For protein analysis, cells were lysed with RIPA buffer 48 hours after transfection and Cycloheximide A. B. injection western blots were probed with antibodies targeting OPA1 andβ-actin (Panel C). Multiple bands correspond to 10ug/mL 50ug/mL DMSO different isoforms of OPA1. Data represent average of three independent experiments (* P<0.05 by one-way ANOVA protein productive mRNA ✓The approach allows us to leverage the wild-type allele and can be used compared to“NoASO” group). NT ASO targets unrelated gene. A X B to potentially treat ADOA in a mutation-independent manner Exon A Exon X Exon B ASO-14 increases OPA1 expression in an OPA1 haploinsufficient *PTC +/- (OPA1 ) cell line Ongoing work protein Non-productive mRNA A B X +/- +/- A. Characterization of OPA1 HEK293 cell line Figure 6. OPA1 haploinsufficient (OPA1 ) HEK293 cells were generated HEK293 cells +/- Our ongoing work focuses on the following areas: using CRISPR-Cas9 gene editing. Similar to ADOA patient cells, OPA1 1.2 1.2 OPA1 mRNA OPA1 protein Figure 2. Novel NMD exon inclusion event (Exon X) identified in the OPA1 gene which leads to the introduction of a HEK293 cells show approximately 50% mRNA and protein levels of that 1.0 1.0 premature termination codon (PTC) resulting in a non-productive mRNA transcript degraded by non-sense mediated +/+ +/- 1. Development of in vitro assays to assess mitochondria function upon ASO observed in OPA1 cells (Panel A). OPA1 HEK293 cells were transfected decay (NMD) (Panel A). As NMD is a translation-dependent process, the protein synthesis inhibitor cycloheximide 0.8 0.8 with different doses of ASO-14, total protein was isolated 72 hours after treatment (CHX) was used to evaluate the true abundance of the event. Panel B shows an increase in OPA1 transcripts 0.6 0.6 transfection. Western blots were probed with antibodies targeting OPA1 containing the NMD exon in HEK293 cells with increasing CHX dose. Other ocular cell lines also validated for the ▪ High resolution imaging to characterize mitochondrial morphology andβ-tubulin (Panel B, quantification in Panel C). Data represent average of 0.4 0.4 presence of the NMD exon (ARPE-19, Y79). two independent experiments (* P<0.05 by one-way ANOVA compared to 0.2 0.2 ▪ Mitochondrial bioenergetics assays to measure function +/- “NoASO” group). ASO-14 increases OPA1 protein levels in OPA1 0.0 0.0 OPA1 NMD event is conserved in the primate and rabbit eye +/+ +/- +/+ +/- +/- HEK293 cells by 50%, which translates to 75% of wild-type levels. OPA1 OPA1 OPA1 OPA1 2. Development of additional OPA1 in vitro systems +/- P93 P942 Figure 3: RT-PCR data from posterior segment of eye of Effect of ASO-14 on OPA1 protein in OPA1 HEK293 line 30 ▪ ADOA patient fibroblast lines 1.8 (3 months) (2.6yrs) Chlorocebus sabaeus (green monkey) with accompanying * B. C. * 25 * 1.6 +/- +/- OD OS OD OS quantification of NMD exon abundance at 3 months and 2.6 OPA1 ▪ Retinal ganglion cells differentiated from OPA1 iPSCs 1.4 20 A X B years of age (N=1/age). Data represents average of OD and 1.2 ASO-14 15 1.0 OS values for each animal. OD: oculus dextrus (right eye); OS: Disclosures No ASO 1nM 10nM 20nM 40nM 80nM 0.8 10 oculus sinister (left eye); P: post-natal day 0.6 OPA1 5 0.4 AV, SA, AC, KHL, JK, JH, IA and GL are employees and hold equity in Stoke NMD event also conserved in the rabbit retina (See Figure 7, 0.2 A B 0 β-tubulin Panels B and C). 0.0 P93 P942 Therapeutics; ZL and RH received financial support from Stoke Therapeutics 0 1 10 20 40 80 Abundance of event is likely to be higher in vivo, given that NMD is presumed active in the tissue ASO-14 (nM) No ASO ASO-1 ASO-2 ASO-3 ASO-4 ASO-5 ASO-6 ASO-7 ASO-8 ASO-9 ASO-10 ASO-11 ASO-12 ASO-13 ASO-14 ASO-15 ASO-16 ASO-17 ASO-18 No ASO No ASO ASO-1 ASO-2 1nM ASO-3 5nM ASO-4 ASO-5 20nM ASO-6 1nM ASO-7 ASO-8 5nM ASO-9 20nM ASO-10 ASO-11 ASO-12 ASO-13 ASO-14 ASO-15 ASO-16 ASO-17 ASO-18 No ASO 1nM 5nM 20nM 1nM 5nM 20nM No ASO 1nM 5nM 20nM 1nM 5nM 20nM Vehicle Ctrl ASO 12uM Test ASO 4uM Test ASO 12uM Vehicle Ctrl ASO 12uM Tes ASO 4uM t Test ASO 12uM %NMD exon inclusion RelativeOpa1 mRNA expression %NMD exon inclusion %NMD exon inclusion ASO-1 Relative OPA1 protein ASO-2 normalized tob-tubulin ASO-3 ASO-4 ASO-5 RelativeOPA1 mRNA expression ASO-6 ASO-7 ASO-8 ASO-9 RelativeOPA1 mRNA expression ASO-10 ASO-11 ASO-12 Relative OPA1 protein normalized tob-actin ASO-13 Relative OPA1 protein ASO-14 normalized to β-tubulin ASO-15 ASO-16 ASO-17 ASO-18 %NMD exon inclusion Relative OPA1 protein normalized tob-actinExhibit 99.2 Antisense oligonucleotide mediated increase of OPA1 expression using TANGO technology for treatment of autosomal dominant optic atrophy 1 2 1 1 1 1 2 1 1 1 Aditya Venkatesh , Zhiyu Li , Syed Ali , Anne Christiansen , Kian Huat Lim , Jacob Kach , Robert B. Hufnagel , Jeffrey H. Hoger , Isabel Aznarez , Gene Liau 1 2 Stoke Therapeutics, Bedford, MA, Medical Genetics and Ophthalmic Genomics Unit, National Eye Institute, National Institutes of Health, Bethesda, MD Rabbit surrogate ASO decreases non-productive splicing and increases OPA1 Specific ASOs reduce non-productive splicing Background expression in wild-type rabbit retinae following intravitreal injection and increase productive OPA1 mRNA levels in vitro Autosomal dominant optic atrophy (ADOA) is one of the most commonly Final conc. Dose A. Study Design A. Dose Dose and Matrices RT-PCR for non-productive OPA1 mRNA Group N Treatment expected in volume diagnosed optic neuropathies. This optic nerve disease is associated with (ug/eye) regimen collected (OU) vitreous* (uM) (uL/eye) Day 1 No ASO 1 3 Vehicle (PBS) NA N/A structural and functional mitochondrial deficits that lead to degeneration Retinae split Single intravitreal injection of along the 2 3 NT ASO 110 12 vehicle/ASO (n=3/group) IVT OU nasal-temporal of the retinal ganglion cells and progressive, irreversible loss of vision. A Test ASO – 30 on Day 1 axes, temporal 3 3 39 4 Female NZW rabbits A X B Low Dose half used for majority of ADOA patients carry mutations in OPA1 and most mutations RNA, nasal for Test ASO – Day 16 4 3 116 12 protein High Dose A B Euthanize animals and collect retinal tissue lead to haploinsufficiency (Lenaers G. et al. Orphanet J Rare Dis 2012). B. Non-productive OPA1 mRNA C. OPA1 encodes a mitochondrial GTPase with a critical role in mitochondrial B. Non-productive OPA1 mRNA Quantification C. Productive OPA1 mRNA – Taqman qPCR Test ASO Test ASO NT ASO Non-productive OPA1 mRNA OPA1 protein Vehicle (PBS) 4uM 12uM 12uM 1.4 * fusion, ATP synthesis and apoptosis. Currently, there is no approved 16 25 1.4 U499 U514 U516 U501 U505 U524 U517 U518 U520 U503 U513 U521 1.2 14 OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS OD OS 1.2 20 1.0 disease-modifying treatment for ADOA patients. Here, we employ TANGO 12 1.0 15 0.8 10 A X B 0.8 (Targeted Augmentation of Nuclear Gene Output), a novel therapeutic 0.6 8 10 * 0.6 0.4 6 0.4 A B 5 approach, that uses antisense oligonucleotides (ASOs), to increase the 4 0.2 * 0.2 2 0.0 0 endogenous expression of OPA1. 0.0 0 OPA1 protein Figure 1: Mechanism of TANGO: OPA1 TANGO reduces non-productive Figure 4. For in vitro screening, ASOs were transfected at 80nM dose into HEK293 cells using Lipofectamine RNAiMax as messenger RNAs (mRNA), which are a transfection agent. For effect on NMD exon, cells were treated with CHX (50ug/mL, 3 hrs.) 21 hours after transfection. β-actin normally targeted for degradation by RNA was isolated and used for RT-PCR (Panel A with quantification in Panel B). For OPA1 mRNA expression, non- nonsense-mediated mRNA decay (NMD) cycloheximide treated cells were used for Taqman qPCR and mRNA expression of OPA1 was normalized to RPL32. Red Figure 7. Rabbits were used as a surrogate for initial in vivo proof of concept studies to test if our ASO can increase OPA1 as shown in Figure 1. In turn, TANGO arrows highlight ASOs that reduce non-productive splicing and increase OPA1 mRNA expression by at least 20%. expression in the retina following intravitreal injection. Female New Zealand White (NZW) adult rabbits were injected increases productive mRNA and protein. Among these, ASO-14 produces the most increase in OPA1 mRNA (30%) with either vehicle, non-targeting (NT) or test ASO, and animals were euthanized after 15 days to obtain retinal tissue. TANGO specifically increases expression Panel A outlines the study design while Panels B and C depict impact on NMD exon and OPA1 expression (* P<0.05 by ASO-14 decreases non-productive OPA1 mRNA and increases OPA1 of canonical target mRNA and full-length one-way ANOVA compared to Vehicle group). Data show that following intravitreal injection in the rabbit eye, our test protein, only in tissues with endogenous ASO reduces non-productive OPA1 mRNA and increases OPA1 expression in retinal tissue. expression in a dose-dependent manner in vitro gene expression. As these events are OD: oculus dextrus (right eye); OS: oculus sinister (left eye); OU: oculus uterque (both eyes) A. Non-productive OPA1 mRNA B. Productive OPA1 mRNA C. OPA1 protein naturally-occurring, TANGO can *Final concentration in the vitreous calculated assuming vitreal volume in the rabbit as 1.5mL * 1.4 1.4 25 upregulate the wild-type alleles in the * * 1.2 1.2 context of autosomal dominant 20 * Conclusions 1.0 1.0 haploinsufficiency diseases such as 15 0.8 0.8 ADOA. 0.6 0.6 We have validated TANGO as a novel therapeutic approach to address 10 * 0.4 0.4 ADOA caused by OPA1 haploinsufficiency 5 * 0.2 0.2 0.0 0.0 0 Additionally, TANGO offers the following advantages for treating ocular diseases: ✓ Intravitreal injection of ASOs permits diffusion throughout the eye, including retinal neurons ✓We identified ASOs that reduce non-productive OPA1 splicing, increase NT ASO ASO-14 NT ASO ASO-14 NT ASO ASO-14 ✓ Long-term efficacy (>1 year in mouse retina) after single intravitreal injection (Kach et al, productive OPA1 mRNA, and increase OPA1 protein levels ARVO Poster Presentation May 2019) A X B OPA1 +/- ✓ASO-14 increased OPA1 protein up to ~75% of wild-type levels in OPA1 ✓ No specialized formulation or encapsulation required for ASO therapy β-actin ✓ Potential to target large genes not amenable to AAV-based gene therapy HEK293 cells A B ✓The rabbit surrogate ASO increased OPA1 protein in vivo in wild-type Figure 5. HEK293 cells were transfected with different doses of ASO-14 or non-targeting (NT) ASO. RNA was isolated 24 OPA1 non-productive splicing event identification and validation hours after transfection and analyzed for impact on non-productive OPA1 mRNA (Panel A) and OPA1 mRNA expression rabbit retinae and was well tolerated for up to 15 days after intravitreal (Panel B) similar to Figure 4. For protein analysis, cells were lysed with RIPA buffer 48 hours after transfection and Cycloheximide A. B. injection western blots were probed with antibodies targeting OPA1 andβ-actin (Panel C). Multiple bands correspond to 10ug/mL 50ug/mL DMSO different isoforms of OPA1. Data represent average of three independent experiments (* P<0.05 by one-way ANOVA protein productive mRNA ✓The approach allows us to leverage the wild-type allele and can be used compared to“NoASO” group). NT ASO targets unrelated gene. A X B to potentially treat ADOA in a mutation-independent manner Exon A Exon X Exon B ASO-14 increases OPA1 expression in an OPA1 haploinsufficient *PTC +/- (OPA1 ) cell line Ongoing work protein Non-productive mRNA A B X +/- +/- A. Characterization of OPA1 HEK293 cell line Figure 6. OPA1 haploinsufficient (OPA1 ) HEK293 cells were generated HEK293 cells +/- Our ongoing work focuses on the following areas: using CRISPR-Cas9 gene editing. Similar to ADOA patient cells, OPA1 1.2 1.2 OPA1 mRNA OPA1 protein Figure 2. Novel NMD exon inclusion event (Exon X) identified in the OPA1 gene which leads to the introduction of a HEK293 cells show approximately 50% mRNA and protein levels of that 1.0 1.0 premature termination codon (PTC) resulting in a non-productive mRNA transcript degraded by non-sense mediated +/+ +/- 1. Development of in vitro assays to assess mitochondria function upon ASO observed in OPA1 cells (Panel A). OPA1 HEK293 cells were transfected decay (NMD) (Panel A). As NMD is a translation-dependent process, the protein synthesis inhibitor cycloheximide 0.8 0.8 with different doses of ASO-14, total protein was isolated 72 hours after treatment (CHX) was used to evaluate the true abundance of the event. Panel B shows an increase in OPA1 transcripts 0.6 0.6 transfection. Western blots were probed with antibodies targeting OPA1 containing the NMD exon in HEK293 cells with increasing CHX dose. Other ocular cell lines also validated for the ▪ High resolution imaging to characterize mitochondrial morphology andβ-tubulin (Panel B, quantification in Panel C). Data represent average of 0.4 0.4 presence of the NMD exon (ARPE-19, Y79). two independent experiments (* P<0.05 by one-way ANOVA compared to 0.2 0.2 ▪ Mitochondrial bioenergetics assays to measure function +/- “NoASO” group). ASO-14 increases OPA1 protein levels in OPA1 0.0 0.0 OPA1 NMD event is conserved in the primate and rabbit eye +/+ +/- +/+ +/- +/- HEK293 cells by 50%, which translates to 75% of wild-type levels. OPA1 OPA1 OPA1 OPA1 2. Development of additional OPA1 in vitro systems +/- P93 P942 Figure 3: RT-PCR data from posterior segment of eye of Effect of ASO-14 on OPA1 protein in OPA1 HEK293 line 30 ▪ ADOA patient fibroblast lines 1.8 (3 months) (2.6yrs) Chlorocebus sabaeus (green monkey) with accompanying * B. C. * 25 * 1.6 +/- +/- OD OS OD OS quantification of NMD exon abundance at 3 months and 2.6 OPA1 ▪ Retinal ganglion cells differentiated from OPA1 iPSCs 1.4 20 A X B years of age (N=1/age). Data represents average of OD and 1.2 ASO-14 15 1.0 OS values for each animal. OD: oculus dextrus (right eye); OS: Disclosures No ASO 1nM 10nM 20nM 40nM 80nM 0.8 10 oculus sinister (left eye); P: post-natal day 0.6 OPA1 5 0.4 AV, SA, AC, KHL, JK, JH, IA and GL are employees and hold equity in Stoke NMD event also conserved in the rabbit retina (See Figure 7, 0.2 A B 0 β-tubulin Panels B and C). 0.0 P93 P942 Therapeutics; ZL and RH received financial support from Stoke Therapeutics 0 1 10 20 40 80 Abundance of event is likely to be higher in vivo, given that NMD is presumed active in the tissue ASO-14 (nM) No ASO ASO-1 ASO-2 ASO-3 ASO-4 ASO-5 ASO-6 ASO-7 ASO-8 ASO-9 ASO-10 ASO-11 ASO-12 ASO-13 ASO-14 ASO-15 ASO-16 ASO-17 ASO-18 No ASO No ASO ASO-1 ASO-2 1nM ASO-3 5nM ASO-4 ASO-5 20nM ASO-6 1nM ASO-7 ASO-8 5nM ASO-9 20nM ASO-10 ASO-11 ASO-12 ASO-13 ASO-14 ASO-15 ASO-16 ASO-17 ASO-18 No ASO 1nM 5nM 20nM 1nM 5nM 20nM No ASO 1nM 5nM 20nM 1nM 5nM 20nM Vehicle Ctrl ASO 12uM Test ASO 4uM Test ASO 12uM Vehicle Ctrl ASO 12uM Tes ASO 4uM t Test ASO 12uM %NMD exon inclusion RelativeOpa1 mRNA expression %NMD exon inclusion %NMD exon inclusion ASO-1 Relative OPA1 protein ASO-2 normalized tob-tubulin ASO-3 ASO-4 ASO-5 RelativeOPA1 mRNA expression ASO-6 ASO-7 ASO-8 ASO-9 RelativeOPA1 mRNA expression ASO-10 ASO-11 ASO-12 Relative OPA1 protein normalized tob-actin ASO-13 Relative OPA1 protein ASO-14 normalized to β-tubulin ASO-15 ASO-16 ASO-17 ASO-18 %NMD exon inclusion Relative OPA1 protein normalized tob-actin