The NanoAssemblrTM Platform: Microfluidic based Manufacture of Nanoparticles for Functional and the Development of RNA Therapeutics

James Taylor, Ph.D. CEO & Co-founder Precision NanoSystems, Inc. Nanomedicines Poised to Deliver the Next Wave of Biotech Innovation Nanomedicines are the “FedEx” of Pharmaceuticals

Nanomedicines with molecular “zip codes”

Targeted delivery to disease cells

2 Nanomedicines have Broad Impact Medicine, Medical Research, & Beyond Delivery Payload Applications

MEDICINE GENETIC Genetic Diseases, Cancer siRNA, mRNA, Cardio, CNS, Regen Med, lnRNA, CRISPR, DNA Vaccines, Diagnostics … ….

SMALL MOLECULE RESEARCH

Chemo, Immuno, Antibiotics, Metabolites, Disease Modeling & Imaging agents, Research … Target Validation, Synthetic Bio BIOLOGIC …

Peptides, lipids OTHER APPS mRNA→antibodies … AgBio, Chemical Material Science …

3 The Magic is in the

aqueous organic

ü Laminar flow – controlled mixing

Channel diameter: Rapid & Controlled -1 ~100 µm Mixing ü Rapid mixing (3 ms )

Staggered ü Nanoliter Reaction volumes: ~ 14 nL Herringbone Mixers ü Low energy input, low shear system Mixer design by: Stroock et al., Science 2002 ü Seamless scale-up

Nanoparticles

Exquisite Control Over Manufacturing Process

4 State of the Art Manufacturing: From Microliters to Liters

Clinical Discovery Development Product

0 – 100 μL 1 mL – 15 mL 25 mL – > 100 L

5 Precision NanoSystems, Inc. Revolutionizing Nanomedicine with Microfluidics

ü Proprietary Reagent Kit “Franchise” ü Proprietary Manufacturing Platform

ü Identify abnormal causing disease ü Enable new nanomedicine products

6 NanoAssemblrTM Platform Accelerating Nanomedicine Development

Bench-Scale Robust Conceptual Manufacturing Nanomedicine Nanoparticle Manufacturing Scale-up Nanoparticle Process Product Formulation Process

Pre-Clinical

NanoAssemblr™ Continuous Scale-Up Benchtop Instrument Flow Platform

7 The NanoAssemblr™ Benchtop Instrument

ü Proprietary Microfluidics-based instrument

ü Enables manufacture of novel nanoparticles

ü Prepare 1.5 mL – 15 mL nanoparticles / run NanoAssemblr™ Benchtop Instrument ü Fast (operates at 4 mL/min - 20 mL/min)

ü Prepare > 30 formulations / day

ü Automated & easy-to-use ü Intra- and inter-operator reproducibility

Microfluidic Cartridge

8 A Flexible & Versatile Platform

Nanoparticles Compositions • Emulsions • Liposomes • Solid-Core Lipid Nanoparticles (LNP) • Polymer Nanoparticles • Peptide Nanoparticles, etc.

Therapeutic Cargo • RNA & DNA • Small molecules • Peptides/

9 Rational Nanoparticle Engineering

Control through Manufacturing Process • Mixing Speed via Flow Rate • Flow Rate Ratio • Concentration • Sequence of Components

Control through Nanoparticle Composition • Nanoparticle components • Component ratios • Payload

10 Limit-Size Nanoparticles (lsLNP) Size Matters

1000 nm

100 nm

10 nm 20 nm 80 nm lsLNP Doxil, Ambisome (20 nm – 80 nm) 1 nm (> 80 nm)

11 Manufacture Lipid Core Nanoparticles

POPC

Triolein

12 Nanoemulsion Size Dictated by Manufacturing Process

B POPC/triolein (60/40 mol/mol) 70

60

50

40

30

20 Particle size, nm 10

0 1 2 3 4 5 6 7 8 9 10 Aqueous/ethanol flow rate ratio

Zhigaltsev, I.V. Et al., Langmuir 2012, 28, 3633−3640 13 Nanoemulsion Droplet Size Dictated by Emulsion Composition

90 Actual diameter 80 Theoretical diameter 70

60

50

40

30

Particlenm size, 20

10 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 POPC/Triolein ratio, mol/mol

Zhigaltsev, I.V. Et al., Langmuir 2012, 28, 3633−3640 14 Manufacture of Aqueous Core Lipid Nanoparticles

POPC, DSPC, etc.

Drug

15 Liposome Size Dictated by Manufacturing process

100 DSPC:Chol:DSPE-PEG (55:42:3)

POPC:Chol:DSPE-PEG (55:42:3) 80

60

40 Z-Ave (nm) Z-Ave

Post-Dialysis Data 20 All PDI < 0.09

0 1 2 3 4 Flow Rate Ratio (Aqueous:Organic) “Limit size” Liposomes are dictated by the Flow Rate Ratio

16 Liposome Size Dictated by Lipid Composition POPC:Cholesterol:PEG-DSPE (3%) 50 0.25

0.20 40

0.15 PDI

0.10 30

Z-Ave Z-Ave [nm] 0.05

20 0.00 0 10 25 35 42 Cholesterol content [mol%]

Liposome size and size distribution is dependent on Cholesterol content

17 Manufacture of Limit-Size siRNA Lipid Nanoparticles

Oligo

Cationic Lipid

DSPC

Cholesterol

PEG-Lipid

18 RNA-Lipid Nanoparticle Size Dictated by Manufacturing Process

ChangingsiRNA-LNP (CationicProcess Lipid:DSPC:Cholesterol:PEG ) 100

80

60

40

20 Diameter (nm) Diameter 0 0 4812 16 20 24 Flow Rate (mL / min) RNA-Lipid Nanoparticles Reach “Limit Size” at High Flow Rates

19 Lipid Nanoparticle Size Dictated by Lipid Composition

60 0.10 50 0.08 40 0.06 PDI 30

20 0.04 0.02

Diameter (nm) Diameter 10

0 0.00 1.0 2.5 5.0

PEG-Lipid Content (mol %)

“Limit Size” is Dependent on RNA-Lipid Nanoparticle Composition

20 Size Matters: Smaller LNP for Subcutaneous Delivery

ChangingsiRNA-LNP (CationicProcess Lipid:DSPC:Cholesterol:PEG )

Chen et al. J Controlled Release 2014, in press.

21 Manufacture of Polymer Drug Conjugate Nanoparticles

Hoang et al. Int J Pharm 2014 :471 22 CellaxTM Polymer-Drug conjugates

100nm 100nm

NanoAssemblr TM Vortex

23 Nanoparticle Size Dictated by Polymer Concentration

CellaxTM Polymer-Drug conjugates 120 0.3

100 0.2 PDI

80 0.1 Z-Ave Z-Ave [nm] 60 0.0 4 8 15 20 30 35 concentration [mg/g]

Particle size is dictated by polymer concentration

24 Polymer Nanoparticle Size Distribution Dictated by Manufacturing Process

CellaxTM Polymer-Drug conjugates 0.3

0.2 PDI

0.1

0.0 10 12 14 16 18 20 22 24 26 28 30 32 Flow-Rate [ml/min]

Polydispersity is dependent on flow rate 25 Reproducible

The Standard Deviation in Nanometers from 150 Independent Batches of a Nanomedicine 1.8 Preparation (Particle Size = 76nm)

26 Assessment of the Robustness of the Manufacturing Process

Design of Experiment (DoE) variables – Lipid Concentration – Flow Rate – Mixing Conditions – Lipid:RNA Ratio

Stable Results = Robust Process = Scalable Process

27 Microfluidics Enables “Seamless Scale-Up”

Microfluidic Mixer

Microfluidic Mixer Aqueous (RNA) Buffer Exchange Microfluidic & Mixer Dilution Nanoparticle Solvent Concentration Nanoparticles (Lipids) Microfluidic Mixer Continuous Flow Pumps Microfluidic Mixer

Parallelized Microfluidic Mixers

28 Continuous Flow Scale-up Manufacture of RNA-Lipid Nanoparticles Using Single Mixer

Seamless Transfer of Optimized Manufacturing Parameters

29 Manifold Microfluidic Parallelization Enables RNA-Lipid Nanoparticle Scale-Up Manufacture

12 mL/min 24 mL/min 48 mL/min

Parallelization of Microfluidic Mixers Enables Higher Throughput

30 We are just Scratching the Surface

Molecular Assembly Line

• Step-wise reactions • Targeted medicines • Post-insertion for targeting • Multi-functional agents • Programmable mixing • Bespoke medicines

31 SUB9KITSTM Reagents Helping to Solve the Genomics Bottleneck

32 SUB9KITS™

RNA-Lipid Nanoparticles for Research

Microfluidic Cartridge prepares RNA-LNP @ ~ 100 uL

lipid

RNA Channel Dimensions: ~100 µm

33 SUB9KITS™ Neutral, “Solid-Core” RNA-Lipid Nanoparticles Mimic Endogenous Delivery Systems

Low Density Lipoprotein (LDL): “Endogenous Lipid Nanoparticles”

Molecular model: Neutral, Solid-Core RNA-Lipid Nanoparticles

Wasan K. M . et al. (2008) . Nat. Rev. Drug Disc. 7: 84- 99 34 RNA-Lipid Nanoparticles: The Current Clinical Gold Standard for RNAi Drugs

-40 Patisiran (ALN-TTR02) is Currently in Phase 3 clinical trials for treatment of Transthyretin-Mediated Amyloidosis -20

0

20

40 Treatment (mg/kg) RelativeBaselineto

60 Placebo 0.150 0.050 0.300 80 0.500 Mean % Serum TTR Knockdown Knockdown Mean%Serum TTR 100 siRNA 10 20 30 40 50 Dose Day † Serum TTR levels were measured in separate Phase I study of ALN-PCS, an RNAi therapeutic targeting PCSK9, which uses identical LNP formulation as ALN-TTR02 B.U.Med.Center, July 2012 35 SUB9KITS™ RNA-Lipid Nanoparticles Transfect Cells via Endogenous LDL Uptake Pathway

RNA-Lipid Nanoparticles

LDLR RNA

ApoE Cell

Astrocytes 36 LDL Receptor Expression in Peripheral Tissue Can be Exploited for RNA-LNP Delivery

LDL receptor family members: LDLR, LRP1, VLDLR, ApoER2, LRP4, LRP1B and Megalin 37 SUB9KITS™ > 85% Reduction in PTEN Expression at Picomolar siRNA Concentrations

*** * NS 150

100

50 PTEN Expression PTEN (% Untreated Control) 0 0.1 1 10 • E18 Primary hippocampal neurons (~pure culture) siRNA Dose (ng/mL) • Transfected at DIV 12-14 7.5 1 ng/mL 75 750 • Target : Phosphotensin homolog 1 (PTEN) Untreated 0.1 ng/mL 10 ng/mL Naked siRNA siRNA Dose (pM) • Protein quantified at 48 h post-transfection Control siRNA

38 SUB9KITS™ Well Tolerated with No Observed Toxicity at Tested Concentrations

100

50 LDH (% of control)

0 10 50 400 siRNA Conc. (ng/mL) • E18 Primary hippocampal neurons (~ pure culture) 10 ng/mL75050 ng/mL 3750 30000 • Transfected at DIV 12-14 Untreated 400 ng/mL siRNA Conc. (pM) • Target gene : PTEN Control siRNA • Lactate Dehydrogenase (LDH) Assay • Performed at 48 h post-transfection

39 SUB9KITS™

PTEN Reduced For ≥ 21 Days

*** *** *** *** *** 150 • E18 Primary cortical neurons (mixed culture) • Transfected at DIV 13 • 100 Target gene : PTEN • siRNA conc = 100 ng/mL (7.5 nM) • mRNA quantified at days 1, 3, 6, 10, 21 • In collaboration with Prof. Yu Tian Wang, UBC 50 PTEN Expression PTEN

(% Untreated Control) 0

Day 1 Day 3 Day 6 Day 10 Day 21 Control siRNA Targeting siRNA

40 SUB9KITS™

Reduction in PTEN Gene Expression at DIV ≥ 2

*** *** *** *** ***

• E18 Primary cortical neurons (mixed culture) 100 • Transfected at DIV 2, 6, 9, 13, 16 • Target gene : PTEN • siRNA conc = 100 ng/mL (7.5 nM) • mRNA quantified 72 h post-transfection 50 • In collaboration with Prof. Yu Tian Wang, UBC

PTEN Expression PTEN

(% Untreated Control) 0

Day 2 Day 6 Day 9 Day 13 Day 16 Transfection Day (DIV)

41 SUB9KITS™

Reduced Protein Expression In Vivo for > 15 Days and up to 1 mm from Injection Site

* 1.0 1.0 ** -actin 0.8 0.8

0.6 -actin 0.6 ns 0.4 0.4 *** *** 0.2 PTEN / 0.2 *** 0.0 0.0 PTEN Expression / PTEN Expression 5 10 15 1 - 2 2 - 3 0 - 0.50.5 - 1 Control Control Distance from Time After Injection Tract (mm) Injection (days)

500nL of 5mg siRNA/mL (2.5 μg) 42 SUB9KITS™ Widespread Distribution and Reduced Protein Expression after ICV Administration

Hippocampus Striatum 1.0 1.2

0.8 1.0 0.8

-actin 0.6 -actin 0.6 ** * 0.4 0.4

PTEN / 0.2 PTEN / 0.2 54 54 0.0 0.0

• DiI fluorescence in CA1 region of the PTEN siRNA PTEN siRNA Hippocampus Control siRNA Control siRNA • robust uptake of nanoparticles by neurons in the cell body layer (sp). 4 μL of 5mg siRNA/mL (20 μg )

43 SUB9KITS™ GluN1 Knockdown In Vivo Results in Functional Disruption of NMDAR Synaptic Currents

1.8 1.6 * 1.4 ** 1.2 -actin

1.0 0.8 0.6 0.4 GluN1 / 0.2 636 0.0

Control Luc siRNA GluN1 siRNA 500nL of 5mg siRNA/mL (2.5 μg)

44 SUB9KITS™ mRNA- Lipid Nanoparticles Mediate eGFP Expression in Primary Neurons

eGFP DiI

Draq-5 Overlay

eGFP mRNA 500 ng/mL (37.5 nM); Image taken at 24 h post-transfection 45 Acknowledgements Precision NanoSystems University of British Columbia Aysha Ansari Prof. Pieter Cullis Chen Wan Gesine Heuck Prof. Carl Hansen Alex Leung Tim Leaver Igor Zhigaltzev Justin Lee Kevin Ou Chris Tam Sam Chen Euan Ramsay Tomas Skrinskas Genc Basha Ismail Hafez James Taylor Paulo Lin Anitha Thomas Ontario Institute for Cancer Research Colin Walsh Prof. Shyh-Dar Li Andre Wild Mark Ernsting David Zwaenepoel Bryan Hoang Joseph Bteich

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