Lipid Nanoparticle-Mediated Delivery of
Messenger RNA
Ian MacLachlan, Ph.D.
Executive Vice President and Chief Scientific Officer
1st International mRNA Health Conference
October 24, 2013
Tübingen, Germany
1
Tekmira’s Lipid Nanoparticle Technology
Components
Amino Lipid
Structural Lipid
PEG - Lipid
Nucleic Acid
Jeffs et al. Pharm. Res. 2005.
40-140 nm diameter
2
Tekmira’s Lipid Nanoparticle Technology
Components
Amino Lipid
Structural Lipid
PEG - Lipid
Nucleic Acid
= 100 nm
~90 nm diameter
Jeffs et al. Pharm. Res. 2005.
3
Tekmira’s Lipid Nanoparticle Technology
Components
Amino Lipid
Structural Lipid
PEG - Lipid
Nucleic Acid
= 100 nm
~80 nm diameter
Jeffs et al. Pharm. Res. 2005.
4
LNP Manufacturing Process Flow Diagram
• Simple controlled mixing
process
• No physical particle sizing
step
• Applicable broadly for
nucleic acid encapsulation
• Highly scalable and
reproducible
• Efficient (high
encapsulation)
• CGMP; protected by IP
Jeffs et al. Pharm. Res. 2005.
5
CGMP LNP Manufacturing Has Been Scaled
0.4 g
50 g
100 g
1000 g
Scale/g
Test
Method
0.4
2
2
10
10
100*
1000*
Appearance
Visual
Pass
Pass
Pass
Pass
Pass
Pass
Pass
pH
pH Meter
7.3
7.4
7.4
7.4
7.4
7.4
7.2
siRNA Assay
AX HPLC
2.2
2.0
1.9
2.0
1.9
2.0
2.2
siRNA Purity
AX HPLC
ND
ND
86.7
89.8
88.4
88.8
95.6
siRNA Encapsulation
Fluorometric assay
96
95
94
93
97
95
92
Dynamic Light
Scattering
Particle Size Analysis
79
0.04
78
0.04
81
0.04
78
0.04
81
0.02
78
0.04
80
0.03
Particle Size:
Z-average
Polydispersity
*Demonstration runs
6
CGMP LNP Manufacturing at Tekmira
Particle Formation Skid in Tekmira CGMP Facility
(suitable for 100g+ batches)
Tangential Flow Ultrafiltration Skid
7
Lyophilized LNP Products
Tekmira has developed
lyophilized LNP to:
• Eliminate cold-chain requirements
• Facilitate use in tropical climates
• Allow use of “meta-stable” formulation
components
Current product characteristics:
•
•
•
•
•
Reconstitution time <15 sec.
Particle size <80 nm
>90% encapsulation
Stable at 40°C
Bioequivalent to wet formulation (efficacy, tolerability)
8
Function of Amino Lipids
Formulating:
Low pH, Charged
N
Storage:
pH ~7, Neutral
Endosome:
pH 5 - 6, Charged
Circulation:
pH ~7.4, Neutral
O
H
O
• Ionizable amino head group allows lipid
to take advantage of surrounding pH
• Linoleyl chains prevent tight packing and
promote fusion
9
N+
O
O
10
Lipid Nanoparticles (LNP) – Experience in Man
Seven LNP Products have entered Clinical Development
Product
Company
Phase
Indication
Treated*
Comments
ALN-TTR01
Alnylam
1
Amyloidosis
24
First demonstration of RNAi silencing
of disease causing protein
ALN-TTR02
Alnylam
2
Amyloidosis
13
Potent pharmacodynamic effect
demonstrated, well tolerated
TKM-Ebola
Tekmira
1
Ebola infection
>1
(ongoing)
TKM-ApoB
Tekmira
1
Hyperlipidemia
17
TKM-PLK1
Tekmira
1
Oncology
>23
(ongoing)
Two responders reported (data
collection ongoing)
ALN-PCS
Alnylam
1
Hyperlipidemia
24
Potent pharmacodynamic effect
demonstrated, well tolerated
ALN-VSP
Alnylam
1
Oncology
41
Two patients treated over 1 year
Results not yet reported
Early LNP formulation used
• Numbers include only publically reported data, and do not include placebo controls
• All LNP products have been manufactured by Tekmira
11
Clinical Validation of LNP Technology and RNAi
Alnylam’s TTR Program Using Tekmira’s LNP
Improvements in LNP Formulation Technology
Translate from The Lab to The Clinic
TTR01 Phase 1 Data
TTR02 Phase 1 Data
100
100
80
>10x
60
40
Placebo
20
TTR01 1.0 mg/kg
0
0
5
10
15
Day
20
Mean Serum TTR
Relative to Baseline
120
Mean Serum TTR
Relative to Baseline
120
80
Placebo
60
TTR02 0.3 mg/kg
40
20
25
0
30
XIIIth International Symposium on Amyloidosis, May 2012
0
5
10
15
Day
20
25
30
B.U. Med Center July, 2012
Single LNP
Dose
“Second Generation LNP”
“First Generation LNP”
Both Products Contain the Same TTR siRNA
LNP Formulation Improvements Result in More than 10 Fold Increase in Potency
12
Evolution in LNP Formulation Technology
Ongoing Formulation Improvements Translate from the Lab to the Clinic
LNP
Potency
12000
10000
8000
6000
4000
Lipid Conjugate
Fold Increase in Potency
14000
2012
Tolerability
TI ~1000
Third Generation
MC3
2000
TI ~5
First Gen.
2006
0
2003 2004 2005 2006 2007 2008 2009 2010 2011 2012 2013
TI ~150
Second Generation
2009
0.01
0.1
1
siRNA Dose (mg/kg)
Year
10
Mean Serum TTR Remaining
Relative to Baseline
100
Data Confirms that LNP Formulation
80
NHP
60
Improvements Translate from Non-
Human
40
20
Clinical Species to Man
0
0
Single LNP Dose
13
5
10
15
Day
20
25
30
B.U. Med Center July, 2012
Observations from Clinical Development
Efficacy Observations
Safety Observations
• Multiple POC for RNAi in human
subjects
• Absence of significant hepatotoxicity
• Multiple POC for LNP in human
subjects
• Multiple doses are well tolerated
• Formulation improvements translate
from the lab to the clinic
• Immune mediated effects may be
dose limiting
• PK is maintained in multiple dosing
• Profound knockdown at dose levels
below the inflammatory threshold
LNP enabled RNAi drugs are a viable therapeutic modality, capable of
delivering products with favorable PK and PD characteristics and a
clinically and commercially viable therapeutic index
14
Alternative LNP Payloads
LNP Enable Essentially Any Nucleic Acid Payload
 Small ‘RNA’ Payloads
• Canonical siRNA
• UsiRNA
• MV-RNA
• miRNA
 Small ‘DNA’ Payloads
• Antisense Oligonucleotides
• LNA
 Larger Nucleic Acids
• Plasmid DNA
• Messenger RNA
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Alternative LNP Payloads
LNP Enable Essentially Any Nucleic Acid Payload
 Small ‘RNA’ Payloads
• Canonical siRNA
• UsiRNA
• MV-RNA
• miRNA
 Small ‘DNA’ Payloads
• Antisense Oligonucleotides
• LNA
 Larger Nucleic Acids
• Plasmid DNA
• Messenger RNA
16
mRNA-LNP Formulations:
• Tekmira has developed a broad library that features thousands of lipid
compounds and LNP formulations.
• The formulations described here, while drawing from our experience
with other nucleic acids, are tailored specifically for mRNA.
• Liver LNP – Liver directed formulation for mRNA delivery
• Tumor LNP – Distal tumor directed formulation for mRNA delivery
• LNP described here contain commercially available mRNA, which is:
•
•
•
•
Capped
Polyadenylated
Fully substituted with pseudouridine and 5-methylcytidine
Purified by silica spin column.
17
mRNA-LNP Formulations:
• mRNA are readily encapsulated using LNP methodology.
• Physicochemical characteristics are similar to LNP currently in
the clinic.
• mRNA-LNP stable at 4°C for > several months, measured by:
• Size
• Polydispersity
• mRNA Encapsulation
• No toxicities at the doses described in this work.
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mRNA LNP Activity In Vivo
Enabling Highly Effective mRNA Delivery and Expression
Luciferase Gene Expression in Balb/C Mice
6 & 24 hrs after IV Administration of mLuc mRNA Liver LNP
(0.5 mg/kg, ~10ug)
10000000
mLuc-LNP (6hr)
Luciferase Activity (pg/g organ)
1000000
4 Orders of Magnitude Greater
Than Previously Described* Positive Control
mLuc-LNP (24hr)
TransIT-mLuc (6hr)
100000
Luciferase expression normalized to mass
quantities of recombinant Luc assayed in buffer.
10000
1000
100
10
Liver
Spleen
Lung
Kidney
Heart
* Kariko et al. Mol Ther. 2012 May;20(5):948-53.
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mRNA LNP Activity In Vivo
Linear mRNA Dose Response Over Three Orders of Magnitude
Luciferase Gene Expression in Balb/C Mice Liver
6 hrs after IV Administration of mLuc mRNA Liver LNP
Luciferase Activity (ng/g Liver)
10000
1000
100
10
1
0.1
0.001
~200ng
0.01
0.1
mLuc Dose (mg/kg)
20
1
~20 ug
mRNA LNP Activity In Vivo
mCherry mRNA Delivery and Expression in the Liver – Negative Control
21
mRNA LNP Activity In Vivo
mCherry mRNA Delivery and Expression in the Liver – 24h
22
mRNA LNP Activity In Vivo
mCherry mRNA Delivery and Expression in the Liver – 24h
23
mRNA LNP Activity In Vivo
mCherry mRNA Delivery and Expression in the Liver – 24h
24
mRNA-LNP Delivery to Orthotopic Liver Tumors
Time Course and Biodistribution of Gene Expression in Scid Mice Bearing Orthotopic Hep3B Tumors
Following 0.5 mg/kg (~10ug) of mLuc-Liver LNP (n=5)
Luciferase Activity (pg/g organ)
1000000
100000
10000
1000
100
10
Liver
Tumor
Spleen
6h
24 h
25
48 h
mRNA-LNP Delivery to Orthotopic Liver Tumors
Orthotopic Hep3B Liver Tumors H&E
Liver
Tumour
26
mRNA-LNP Delivery to Orthotopic Liver Tumors
mCherry Expression @ 24h
Liver
Tumour
27
Biodistribution of LNP-mRNA Expression in Tumor-bearing Mice
Distal Tumor Model –Tumor Directed LNP
Luciferase mRNA Expression Following IV Administration of mLuc-Tumor LNP
in SubQ Hep3B Tumor-bearing Scid Mice (n=4, 1.0mg/kg (~20ug) luc mRNA )
Luciferase Activity (pg/g Tissue)
1000000
Liver
Spleen
Lung
Kidney
Heart
Tumor
100000
10000
1000
100
2h
4h
6h
8h
28
16 h
24 h
48 h
LNP-mRNA Expression in Tumor-bearing Mice
Tumor-mRNA Expression Is Long Lived Relative to Other Tissues
Luciferase mRNA Expression Following IV Administration of mLuc-Tumor LNP
in SubQ Hep3B Tumor-bearing Scid Mice (n=4, 1.0mg/kg (~20ug) luc mRNA )
Luciferase Activity (pg/g Tissue)
1000000
100000
Tumour
Liver
Spleen
10000
1000
100
0
5
10
15
20
25
30
Time Point (h)
29
35
40
45
50
Summary
• LNP delivered siRNA are a clinically and commercially viable
technology enabling the development of RNA therapeutics.
• mRNA are readily encapsulated and delivered using LNP.
• Tekmira has designed LNP for hepatic delivery, oncology
applications, inhalation and delivery to immune cells.
• The results suggest a role for LNP in the development of
mRNA therapeutics.
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Acknowledgements
Tekmira
James Heyes
Lorne Palmer
Mark Wood
Amy Lee
Adam Judge
Lloyd Jeffs
Peter Lutwyche
Marjorie Robbins
Sean Semple
Ed Yaworski
Kevin McClintock
Ellen Ambegia
Janet Shaw
Nancy Fuselli
AWARD/CONTRACT
No. W9113M-10-C-0057
31
Lipid Nanoparticle-Mediated Delivery of
Messenger RNA
Ian MacLachlan, Ph.D.
Executive Vice President and Chief Scientific Officer
1st International mRNA Health Conference
October 24, 2013
Tübingen, Germany
32