Low Volume Production Of Nanoparticles That Are Effective

Cell Health Cell Health

Reporter Gene Expression Reporter Gene Expression

Cell Health

Reporter Gene Expression

6 30 54 78 102 126 150 174 198 222 246 270 294 3180.0

0.1

0.2

0.3

0.4

0.5

Hours Post Plasmid LNP Treatment

Neurite Length (mm) / Cell Body Cluster

Representative ImagesPhase - Formulation No.4 @ 78h

GFP Fluorescence - Formulation No.4 @ 78h



Untreated

Formulation 1

Formulation 3Formulation 4

Formulation 2

Untreated

Formulation 1


Formulation 2

Untreated

Formulation 1


Formulation 2

Untreated

Formulation 1


Formulation 2

Untreated

Formulation 1


Formulation 2

Untreated

Formulation 1


Formulation 2

6 30 54 78 102 126 150 174 198 222 246 270 294 3180

25

50

75

100


% of phase cell bodies that are GFP Positive



0

40

80

120

160

0

40

80

120

160

0.0

0.2

0.4

0.6

0.8

1.0n.s.

n.s.

0.0

0.2

0.4

0.6

0.8

1.0

1.5 3 4PEG-DMG (mol %)

Diameter, Z. Avg (nm) PDI

Encapsulation Efficiency (%)


4 6 8N/P ratio

n.s.

n.s.

Lipid Composition CL:DSPC:Cholesterol:PEG-DMG50:10:48.5:1.5 mol%CL = cationic lipid as labeled on x-axis

Init. Lipid Conc. 50 mM

mRNA GFP (996 nt), 0.025 mg

N/P Ratio 4

Organic Solvent Ethanol

Aqueous Phase 0.695 mg/mL mRNA in 100mMSodium Acetate

Instrument Setting 3x

Downstream Processing 4x Dilution in nuclease-free PBS

Lipid Composition PNI-ILa:DSPC:Cholesterol:PEG-DMG 50:10:38.5:1.5 mol%


N/P Ratio

mRNA Mass (mg) Conc. (mg/mL)

GFP (996nt)

Aqueous Phase mRNA in 100 mM Sodium Acetate




1.5 3 4


0

20

40

60

80

100

PEG-DMG (mol %)

n.s.

0.0

0.2

0.4

0.6

0.8

1.0



0

50

100

150

200

DOTAP DOTMAIonizable/cationic lipid

n.s.

0

20

40

60

80

100

PNI-ILa

DOTAP DOTMA PNI-ILaIonizable/cationic lipid

4 6 80.0

20.0

40.0

60.0

80.0

100.0

N/P ratio

4 6 8

0.025 0.0167 0.01250.695 0.464 0.347

6 18 30 42 54 66 78 90 102 114 126 138 150 1620.0

0.2

0.4

0.6

0.8


6 18 30 42 54 66 78 90 102 114 126 138 150 1620

20

40

60


6 18 30 42 54 66 78 90 102 114 126 138 150 1620.0

0.2

0.4

0.6

0.8


6 18 30 42 54 66 78 90 102 114 126 138 150 1620

20

40

60


Cell Type iPSC-Derived NPCs

N/P Ratio 6

Payload GFP Plasmid

Treatment Point Day 1 of differentiation protocol

Dose 0.6 µg DNA /mL media

Apo-E 1 µg/mL media

Biological Assay Live-cell imaging:Phase: Neurite LengthFluorescence: GFP Expr.

Capture Frequency 6 Hours

Total Observed Period

21 Days

Representative ImagesPhase - Formulation No.3 @ 96h

GFP Fluorescence - Formulation No.3 @ 96h

Cell Type iPSC-Derived NPCs

N/P Ratio 4

Payload GFP Plasmid

Treatment Point Day 14 of differentiation

Dose 0.6 µg DNA /mL media

Apo-E 1 µg/mL media

Biological Assay Live-cell imaging:Phase: Neurite LengthFluorescence: GFP Expr.

Capture Frequency 6 Hours

Total Observed Period 7 Days

1Precision NanoSystems Inc., Vancouver, BC, Canada, 2International Collaboration on Repair Discoveries (ICORD), 3Department of Zoology, 4Stem Cell Program and Institute for Regenerative Cures, University of California Davis Health Systems, Sacramento, CA, USA, 5Genome Center, MIND Institute, and Biochemistry and Molecular Medicine, University of California, Davis, CA, USA

• Nucleic acid therapies permit access to previously undruggable pathways to treat or potentially cure a wide range of diseases, and lipid nanoparticles (LNPs) are the most clinically advanced non-viral delivery systems

• Significant need exists for robust manufacturing of LNPs at all scales, particularly at very low volumes to enable efficient screening of lipid components and nucleic acid payloads at earlier stages of drug development

• Here, we demonstrate the use of a low volume microfluidic device that provides consistent, controlled formulation conditions for encapsulating just tens of micrograms of mRNA and plasmids into LNPs

• LNP materials were systematically screened for size, polydispersity index, and activity in clinically relevant induced pluripotent stem cell (iPSC)-derived neuroprogenitor cells (NPCs) and cortical neurons

25-250 µL 1 - 15 mL 10 - 1000 mL 24L in 4.5h

NanoAssemblr™ Systems Span all Scales

Gene Delivery by Lipid Nanoparticle (LNP)

Spark™ Benchtop Blaze™ 8x Scale-Up

Systems employed in this study

Microfludic Nanoparticle Production

The NanoAssemblr™ platform uses microfluidics to precisely control the manufacturing of nanoparticles from microlitre to litre scales.

The NanoAssemblr Spark Workflow

• Uptake of LNPs occurs via low density lipoprotein receptor mediated endocytosis. Ionizable cationic lipids mediate endosomal disruption and release of nucleic acid

• LNPs were tested for expression in clinically relevant iPSC-derived NPCs and cortical neurons

• Formulations were rapidly produced with minimum consumption of valuable mRNA and plasmid

• Formulation volumes were < 100µL and required only 12-25 µg of nucleic acid

• Formulations were produced in sterile hood and immediately characterized or applied directly to iPSC-derived cells in culture

!"#"

!#"#"#"

#"

!

!

!

#"

#"

Nucleic Acid-LNP

Uptake

Nucleic AcidReleased

pH Change

Cytoplasm

Cell Membrane

Highest PEG concentration reduces encapsulation efficiency

Ionizable cationic lipid produces LNPs with lower size and PDI

Encapsulation efficiency and size reach threshold at N/P ≥ 6

Size measured by DLS and Encapsulation Efficiency by Ribogreen assay. Values represent the mean of triplicate formulations. Error bars are the standard deviation. Means grouped by a horizontal line are not significantly different (Tukey’s multiple comparison test, P>0.05).

Further Details

Size measured by DLS and Encapsulation Efficiency by Ribogreen assay. Values represent the mean of triplicate formulations. Error bars are the standard deviation and are not drawn when error is less than the size of the symbol. Means grouped by a horizontal line are not significantly different (Tukey’s multiple comparison test, P>0.05).

Further Details

Size measured by DLS and Encapsulation Efficiency by Ribogreen assay. Values represent the mean of triplicate formulations. Error bars are the standard deviation and are not drawn when error is less than the size of the symbol. Means grouped by a horizontal line are not significantly different (Tukey’s multiple comparison test, P>0.05).

Further Details

Lipid Composition PNI-ILa:DSPC:Cholesterol:PEG-DMG50:10:40-x:x | x = 1.5, 3, 5 mol%


mRNA GFP (996 nt), 0.025 mg

N/P Ratio 4


Aqueous Phase 0.695 mg/mL mRNA in 100mMSodium Acetate



Formulation Details

Formulation Details

Formulation Details

Formulations with similar properties perform differently in iPSC-derrived NPCs undergoing differentiation: Biological activity is a necessary screen

A

Systematic screening of plasmid LNP formulations in mature human iPSC-derived cortical neurons: N/P ratio had a profound impact

N/P 6: Impacts Cell Health and Has Low Reporter Expression

B N/P 4: No Impact on Cell Health and Has Strong Expression of Reporter Gene

Treatment Details

• Formulation composition affects neuronal development: formulations 1 and 2 exhibit large decrease in neurite length that is not recovered over time

• Reporter gene expression peaks ~78h after treatment and appreciable expression is still present 14d following treatment

• The same formulations that do not impact neurite length also display high GFP expression, with approximately 80% of cell bodies expressing GFP by 78 hours

Treatment Details

• The N/P ratio is important to formulation efficacy for plasmid delivery to human iPSC-derived cortical neurons.

• Different N/P ratios were tested to identify candidate formulations with optimal performance in this therapeutically relevant cell type.

B Formulations 3 & 4 Exhibit the Least Impact on Neurite Health and The Highest Reporter Gene Expression

1Formulation Number

2 3 4

1Formulation Number

2 3 4

0

50

100

Encapsulation Effiency N/P 6N/P 4

Diameter, Z. Avg (nm)N/P 6 N/P 6

PDI

N/P 4N/P 4

0

100

200

0.0

0.2

0.4

A Physical properties similar between formulations with different lipids and N/P ratios

Further Details

• A panel of formulations containing PNI-ILa with different helper lipids was created at two N/P ratios

• Formulations 2, 3, and 4 have similar properties

• Panel was created rapidly with the Spark at µg quantities with encapsulation efficiency > 80%

Size measured by DLS and Encapsulation Efficiency by Ribogreen assay. Values represent the mean of duplicate formulations. Error bars are the standard deviation and are not drawn where error is smaller than the symbol.

1. Pipette Reagents In

2. Cap 3. Insert

4. Push “Start” 5. Pipette Particles Out

6. Dilute

Ready!

Characterizeor

Apply Directlyto Cells<10s

a cb

Buffer +mRNA

mRNA-LNP

Ethanol +lipids

LaminarMixing in < 1 ms

1

2

3

Purpose and Objectives Screening microgram mRNA formulations

Methods

Case Study: Optimizing LNP formulations for plasmid expression in iPSCs

Conclusions

• Microliter formulations containing microgram quantities of mRNA were rapidly produced using the NanoAssemblr Spark microfluidic system to systematically screen compositions against properties and activity

• PEG-lipid content ≥ 4 mol% reduced encapsulation efficiency; PNI-ILa produced LNPs with more favourable size and PDI than other cationic lipids; N/P ratios > 6 did not appreciably affect properties

• A panel of 4 formulations formulated at N/P 6 and N/P 4 were rapidly developed for testing in iPSC-derived neurons

• On iPSC-derived NPCs, formulations 3 and 4 outperformed 1 and 2 in terms of cell tolerance and reporter gene expression

• On iPSC-derived cortical neurons, all formulations at N/P 4 were better tolerated than N/P 6, and formulations 3 and 4 at N/P = 4 were most effective in inducing reporter gene expression

• Despite similar phsyical properties, formulations had very different in vitro efficacy• The Spark is ideal for efficient screening of formulations with valuable payloads in

clinically relevant cell types

/company/precision-nanosystems-inc @PrecisionNanowww.precisionnanosystems.com [email protected]

Low Volume Production Of Nanoparticles That Are Effective Transfection Systems In iPSC-derived Cells, Immune Cells and Other Primary Cell CulturesAndrew Brown, Chelsea Cayabyab, Rebecca A. G. De Souza, Tara Fernandez, Grace Tharmarajah, Anitha Thomas, Samuel Clarke and Euan Ramsay Precision NanoSystems Inc, Vancouver, BC, Canada

This poster is interactve!Download the free “layar” app on your

smartphone or tablet and use it to scan this poster. Enriched features are indicated with this logo.

Detailed Materials & Methods: Scan with Layar app.

Time lapse video of iPSC-NPCs growing and expressing GFP over 21 day period: Scan with Layar app.

Time lapse video of iPSC-derived cortical neurons growing and expressing GFP over 6 day period: Scan with Layar app.

Documents

Low Volume Production Of Nanoparticles That Are Effective