The NanoAssemblrTM Platform: Microfluidic based Manufacture of Nanoparticles for Functional Genomics 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, Molecular Biology 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 Microfluidics
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 genes 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/proteins
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 Protein 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 gene : 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 Gene Expression 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