October 8, 2018

John Tilton, MDCenter for Proteomics and Bioinformatics

Mitchell Drumm, PhDDepartment of Genetics

Jeff Coller, PhDCenter for RNA Molecular Biology

Protein and RNA Therapeutics for the Treatment of Lung Diseases

• Platform technology for protein and RNA therapeutics for targeted in vivo delivery.

• Validated delivery of CRISPR-Cas9:gRNA complexes, Crerecombinase, enzymes, antigens, and transcriptional regulators.

• Compatibility with mRNA and suppressor tRNA technologies.

• Powerful imaging technologies for whole-body analysis of on-and off-target delivery.

• Comprehensive reporter and disease models to evaluate efficacy of correction or read-through of nonsense mutations.

• Flexible technologies that can be modulated and repurposed for other genetic diseases.

Key Technology Attributes

Market Need

• Virtually all current protein therapeutics act at the cell surface or outside of cells due to the lack of an effective platform for intracellular delivery.

• Similarly, RNA therapeutics are primarily limited by an effective intracellular delivery platform.

• As proof-of-concept, we are focusing on nonsense mutations in cystic fibrosis, which affect a comparable number of patients as those targeted by Kalydeco. • 70/71 CF nonsense mutations are not treated by current

drugs.

Protein & RNA Delivery Platform• We are developing a platform for targeted, intracellular

delivery of protein and RNA to tissues in vivo.o Efficient delivery to mammalian cellso Engineered to improve safety and reduce immunogenicityo Mimics viral biology for in vivo delivery

• nanoscale PrOtein Delivery (nanoPOD) platform

Efficient delivery of cargo to cells

Protein delivery (β-lac.)Gene delivery (EGFP)

• Lentiviruses are far more efficient at delivering protein cargos than DNA.

• We have delivered Cre recombinase, Cas9:guide RNA ribonucleoproteincomplexes, enzymes, antigens, and transcriptional activators.

Cre delivery

mock Cre nanoparticles

Cas9:gRNA delivery

200bp-

500bp-

Rationally engineered for safety & efficacy• We have eliminated the ‘genome’

and many structural proteins.

• We have introduced specific mutations into the incorporation protein (Vpr) to eliminate undesirable effects.

• Optimization of cargo loading with minimal Vpr regions and KO of wild-type Vpr.

• Protease cleavage site optimization for separation of cargo from Vpr.

G2 cell cycle arrest

RNA electroporation of immune cells with nanoPOD components and homing receptors.Particles protected from serum components and macrophages.Immune cells migrate into tissues, directed by receptors (e.g. CCR4 for lung). Particles carried through extracellular matrix to target cells.Cells produce nanoPODs within the local microenvironment.

Harnessing biology for in vivo delivery• Lentiviruses spread throughout individuals within immune cells

that migrate to tissues and release viruses. This protects them from numerous in vivo barriers to nanoparticles.

Cell-free delivery

In vitroIn vivo

Cell-associated delivery

RNA delivery using nanoPODs• RNA incorporation strategies

MS2-Vpr incorporation

Integration defective LV

• Suppressor tRNA delivery enables read-through of premature termination codons (PTCs):

0%

5%

10%

15%

20%

0 100 200 400

Perc

ent r

ead-

thro

ugh

Suppressor tRNA Transfected

Supporting Technologies: Imaging of Delivery

• We have powerful imaging platforms to guide delivery strategies.

RosamT/mG mice

mock Cre nanoparticles

Primary mouse hepatocytes

RosamT/mV-akaLuc mice

Flow cytometry

Harvest organs

Dissociate cells

Quantify deliveryefficiency

(RFPmVenus)

Bioluminescence imaging

Kinetics, low-resolution

biodistribution

Cryo-fluorescence imagingHigh-resolution biodistirubtion

Supporting Technologies: Cellular & Animal Models

• We also have powerful cell and animal models of human genetic diseases, with a particular emphasis on cystic fibrosis.

Cell models Organoid models Mouse models

Reporters with patient-specific mutations

Disease models with patient-specific mutations

Technology Needs• We have two primary goals to move this technology

forward:

• Demonstration of in vivo delivery of cargo to lung and other tissues.

• Adaptation of mRNA and suppressor tRNA cargos to nanoPODs.

Future Project Plan

• Aim 1: Deliver cargo to lung and GI tissues in vivo.• Cell-free particle approach• Cell-associated (Trojan Horse) approach

• Aim 2: Adapt mRNA cargos to nanoPODs• MS2-Vpr coat tags• Integration defective lentiviruses

Project Milestones

• Delivery of Cre cargo to lung and GI epithelial cells with ≥ 3% of cells undergoing red-yellow conversion

• Successful delivery of tRNA and mRNA cargo in vitro

Pending Funding

• $300K Falk Catalyst Award pending

• CAHH funded work will help support additional application for a $1M Falk Transformation Award to test efficacy of nanoPOD

Market Opportunity

Daria Fedyukina, PhDTranslational Officer, CWRU SOM

CF is caused by PTCs in >2K US patients and no disease-modifying treatments are available

~2,000 mutations in CF patientsF508del/F508del 26 12

Kalydeco

Symdeko

70 PTC Other mutations

No disease-modifying treatments

30k CF patients in the U.S.

36%

6.9% PTC3.9%3.8%

50%

Kalydeco - $845M in 2017

UGA1,290 patients

UAG420 patients

UAA360 patients

2.1k CF patients w/PTC in the U.S.

Note: Orkambi (lumacaftor/ivacaftor) is the 3rd drug of current standard of care. It is indicated only for homozygous F508del patients. It is being gradually substituted with Symdeko (more efficacious) since Feb 2018 in all patients except in those of age 2-6 and those whose medical insurance does not reimburse more expensive Symdeko (delta $30k per patient p.a.); Source: Vertex 2017 Annual report; www.CFTR2.org database; FDA Labels of Kalydeco and Symdeko; Castellani et al, J Cystic Fibrosis, 2018; Ponzano et al, Front Clin Pract CF, 2018; Wiencek et al, Clin Chem, 2018; Martinovich et al, Mol Cell Pediatr, 2018; Interviews w/ CF KOLs

Clinical pipeline focused on F508del patients. An mRNA candidate targeting certain PTCs is in early stage of development.

~2,000 mutations in CF patientsF508del/F508del2612

Kalydeco

Symdeko

70 PTCOther mutations

No disease-modifying treatments

F508del heteroz.

VX-445, VX-659 (Ph3);GLPG2451** (Ph1)

~70-80% CF patients

Clinical pipeline*

PTI-428 (Ph2);QR-010 (AO***, Ph1/2);PTI-801, PTI-808 (Ph1);

~50% CF patientsMRT5005

(mRNA LNP, Ph1/2)<57% CF patients

Both alleles w/Class 1 or 2 mut.Both alleles w/

Class 1 or 2 mut.

NanoPOD: UGA-tRNA

Note: LNP = liposomal nanoparticles; *Excluded: trials of approved drugs, antimicrobial agents, devices, procedures, trials by academic institutions without commercial partner; **Heteroz.F508del only if non-responsive to potentiators; ***AO editing, changes A back to I/G, for G-to-A mutations, delivered IV, in liposomes, as a single dose (inhalation) or a short course; Source: ClinicalTrials.gov; company websites; press releases; www.CFTR2.orgdatabase; FDA Labels of Kalydeco and Symdeko; Castellani et al, J Cystic Fibrosis, 2018; Ponzano et al, Front Clin Pract CF, 2018; Wiencek et al, Clin Chem, 2018; Martinovich et al, Mol Cell Pediatr, 2018; Interviews w/ CF KOLs

NanoPOD-tRNA is differentiated from pipeline mRNA and AO candidates

Parameter mRNA AO NanoPOD-tRNA

Delivery vehicle Liposomes Liposomes Lentiviral capsid

Intracellular delivery Yes Yes Yes

RoA Inhaled Inhaled IV

Half life Short Short Medium

Targeted mutations All at once One at a time Several at a time

Editing No Yes No

Utility in other lung disorders w/o modifications

No No Yes

Multi-gene diseases Poor fit Poor fit Good fit

Autosomal dominant diseases

Poor fit Good fit Good fit

Source: company websites; clinicaltrials.gov

Upon PoC in CF, NanoPOD technology has a potential to address many rare and non-rare lung disorders

Disease examples Inheritance PTC Unmet need

A1AT (orphan) AR Yes High

PCD (orphan) AR >20% High

Surfactant dysfunction (ultra-orphan)

AR: SFTPB & ABCA3;AD: SFTPC

Yes High

NanoPODtechnology

for lung disorders

Delivery of mRNA or

tRNA

Delivery of known

therapeutics (proteins or their mRNA)

Examples Biologic treatment Unmet need

Severe asthma reslizumab, omalizumab, mepolizumab,

Better distribution in the lung

Alveolar proteinosis

GM-CSF

Some autoimmune/ rheumatologic diseases with pulmonary phenotype

Note: A1AT = alpha-1-antitrypsin deficiency; PCD = primary ciliary dyskinesia;PCD is caused by mutations in over 30 genes. PTC mutations have been reported for several genes; Source: OMIM; orphan.net; GHR; webMD; interviews w/ pulmonologists; Bukowy-Bieryllo et al, RNA Biology, 2016; van Haasteren et al, Expert Opin Biol Ther, 2018; Wert et al, Pediatr Dev Pathol, 2009; ChILD Foundation; PCD foundation;

Thank you

Appendix

CF mutations are classified according to mechanism of disruption of CFTR production and function

Mutation class Resulting CFTR protein defects Specific

mutations Frequency Phenotype Approved drugs

1 • No functional protein is synthesized • PTC 2-5% Severe None

2• Full protein, but misfolded• Does not reach the apical cell surface

• F508del 70-90% Severe

Orkambi(lumacaftor/ivacaftor)

Symdeko(tezacaftor/ivacaftor)

3

• Full-length CFTR protein• Reached the apical cell surface, but

exhibits abnormal gating (channel persists in the closed state)

• G551D, G1349D, etc.

4-5% Variable Kalydeco (ivacaftor)

4

• Full-length CFTR protein• Reached the apical cell surface, but

transport of Cl- is reduced due to channel pore defect (impaired conductance)

• R117H, R334W, R347P, etc.

N/A Variable Kalydeco (ivacaftor)

5• Reduced number of CFTR transcripts due

to mutations that cause mRNA mis-splicing or interfere with the promoter activity

• 2789+5G>A, 3849+10kbC>T

N/A Variable N/A

6

• Reduced CFTR stability at the cell surface• C-terminal mutations result in accelerated

turnover (channel removal from the plasma membrane)

• 4326delTC, 4279insA, etc.

N/A variable N/A

Note: PTC = premature termination codons; Ivacaftor = CFTR potentiator: Potentiates the channel open probability (or gating) of CFTR protein located at the cell surface; Lumacaftor/ Tezacaftor = CFTR corrector: Improves delivery of CFTR (normal and mutant forms) to the cell surface; Source: Ponzano et al, Front Clin Pract CF, 2018; FDA labels; Wiencek et al, Clin Chem, 2018;

Appendix

71 PTC represents 6.9% of all CF patients. Each of three stop codons causes CF ~1-4% of patients.

0.0%

0.5%

1.0%

1.5%

2.0%

2.5%

3.0%

G54

2XW

1282

XR

553X

R11

62X

E60X

Q49

3XY1

092X

R11

58X

E585

XR

75X

Y122

XQ

220X

W10

89X

R70

9XS4

89X

W84

6XK7

10X

Q39

XQ

890X

S466

XE9

2XQ

552X

L732

XR

785X

R76

4XE8

31X

Q13

13X

W12

04X

R85

1XL1

254X

Q98

XG

330X

E110

4XE8

22X

S119

6XE1

371X

E193

XS9

12X

S125

5XW

401X S4X

W57

XC

276X

R79

2XY8

49X

Y275

XG

673X

Y913

XG

27X

W10

98X

C52

4XQ

525X

L88X

Q14

11X

G55

0XQ

1042

XW

1145

XQ

2XQ

414X

R11

02X

Q13

82X

W19

XQ

685X

W88

2XQ

1412

XQ

30X

W21

6XW

496X

Q71

5XQ

720X

Q13

30X

Frequency of 71 PTC mutations in CF patients

Total frequency of PTCs in CF = 6.9% All PTC mutations lead to severe phenotype Only E831X (splice site mutation) is addressed by current SoC

E831X is targeted by Kalydeco and

Symdeko

1.2% 1.4%

4.3%

0.0%

1.5%

3.0%

4.5%

6.0%

UAA UAG UGA

6.9% CF PTC patients by stop codon

Source: www.CFTR2.org database; FDA Labels of Kalydeco and Symdeko; interviews with CF experts;

Appendix

In the clinical pipeline, RNA therapeutic present the strongest competitive threat to NanoPOD-tRNA

Phase 1 Phase 1/2 Phase 2 Phase 2/3 or 3

Pipeline of disease-modifying drug candidates*

Small molecule

RNA

Peptide

Specifically includes PTC

Other mutationsGenetics agnostic

Molecule type Addressable patients

MRT5005 (inhaled)CFTR mRNA in nanoparticles

Both alleles w/Class 1 or 2 mut.Translate Bio

VX-445** combo w/Tez-IvaHomoz. & heteroz. F508del

Vertex

QBW276, inhaledNa+ channel blocker, All CF

Novartis

GLPG2451CFTR potentiator; Hom. F508del,

Het. if non-responsive to IvaGalapagos

QR-010 (eluforsen), inhaled single or short courseAO editing, G-to-A mut.,

Homoz. F508delProQR Therapeutics

fenretinideAnti-inflammatory

All CFLaurent Pharma

PTI-801 (combo variations)CFTR corrector; mainly for

homoz.F508delProteostasis Therapeutics

PTI-808 (combo variations)CFTR potentiator; mainly for

homoz.F508delProteostasis Therapeutics

lenabasumSelective cannabinoid receptor

type 2 agonist. All CFCorbus Pharma

PTI-428CFTR amplifier

Homoz. F508delProteostasis Therapeutics

RPL554, inhaledPDE-3i/ PDE-4i

All CFVerona Pharma

SPX-101ENaC regulatory peptide

All CFSpyryx Biosciences

VX-659** combo w/Tez-IvaHomoz. & heteroz. F508del

Vertex

• Recently, Ph3 ataluren trial failed. Ataluren was meant to promote read-through of PTC

• ProQR Therapeutics is also conducting preclinical/discovery studies of 3 AO candidates targeting most frequent PTC mutations in CF:

• QRX-042 for W1282X (2.5%)• QRX-036 for G542X (1.2%)• QRX-052 for R553X (0.9%)

Note: *Excluded: trials of approved drugs, antimicrobial agents, devices, procedures, trials by academic institutions without commercial partner;**Vertex will select VX-445 or VX-659 for marketing and launch, based on Ph3 clinical trial results; Source: ClinicalTrials.gov; company websites; press releases; interviews with CF experts

Appendix