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Non-viral Delivery of ZFN mRNA Enables Highly Efficient In Vivo Genome Editing of Multiple Therapeutic Gene Targets
Anthony Conway, Ph.D.
May 19, 2018
2
Engineered zinc finger nuclease (ZFN) technology
Lipid nanoparticles (LNP) for non-viral delivery of ZFN mRNA
Targeting the liver for potential therapeutic applications
• Single gene knockout
• In Vivo Protein Replacement
Targeting lung epithelial cells via LNP
Outline
ZFNs enable genome editing
3
Non-Homologous End Joining (NHEJ)
Homologous Recombination
DNA Donor with Homology
Gene Knockout Targeted Integration
Double Strand Break
Insertion
Deletion
Alternative to AAV Delivery
Lipid nanoparticle (LNP) technology for efficient in vivo non-viral delivery of ZFN mRNA to hepatocytes
4
• No pre-existing immunity • Transient mRNA delivery/expression• Enables repeat dosing
ApoE facilitates receptor binding, endocytosis in hepatocytes
Endosomal escape of mRNA by amino lipid protonation
~80 nm
ApoE binds after loss of PEG-lipid
P < 0.0001
P < 0.0001
ZFN mRNA delivery via LNP allows for accumulation of genome modification within the murine liver following repeat administration
5
Linear regression analysis
Progressive increase in indels out to 6 repeat doses
mRNA-LNP primarily traffics to liver cells
6*Residual nucleated blood cells lower overall genome editing fraction in unperfused bulk liver
Genome editing highly targeted to liver
LNP PBS
Systemic delivery of liver-targeted ZFP therapeutics via mRNA-LNP for single gene knockout applications
7
LNP PackagingRepeat
AdministrationProgressive
Gene Knockout
Lipids
ZFN mRNA
Targets IncludeTTR, PCSK9
1 2 3 4
Ind
els
(%
)
Pro
tein
Exp
ressio
n
Doses
Therapeutic
Level
0.8 mg/kg dose
60% gene knockout in bulk liver tissue
ZFNs targeting murine TTR delivered via mRNA-LNP in mice achieves >90% gene disruption from the liver
8
90% TTR protein knockdown in plasma
60%
90%
No elevated liver toxicity
4-Fold Lower Dose
Optimized ZFNs targeting murine TTR results in highly efficient gene disruption at very low mRNA-LNP doses with no signs of toxicity
9
>60% gene knockout in bulk liver tissue
ALT AST
ZFNs targeting murine PCSK9 delivered as mRNA-LNP also achieved>90% protein knockdown in plasma
ZFNs targeting murine PCSK9 delivered via mRNA-LNP achieved >90% protein knockdown from the liver
10
ZFNs enable genome editing
11
Non-Homologous End Joining (NHEJ)
Homologous Recombination
DNA Donor with Homology
Gene Knockout Targeted Integration
Double Strand Break
Insertion
Deletion
Systemic delivery of ZFP Therapeutics via AAV vectors may allow for in vivo correction of monogenic disease
12
Currently Recruiting Patients in U.S.
Hemophilia B (SB-FIX)MPS I (SB-318)MPS II (SB-913)
Lysosomal storage diseases – MPS I and MPS II
13
Accumulation of GAGs (e.g., dermatan and heparan sulfates) in the lysosome of all cells leads to dysfunction in several tissues
Glycoaminoglycans (GAGs)
a-L-Iduronidase(IDUA) in MPS I
Iduronate 2-Sulfatase(IDS) in MPS II
- Cognitive decline
- Organomegaly- Cardiomyopathy- Respiratory complications
- Skeletal and orthopedic issues
- Short stature
- Carpal tunnel- Joint stiffness
*
**
IDS
ac
tivit
y(n
mo
l/h
r/m
L)
IDS
ac
tivit
y(n
mo
l/h
r/m
g)
Mouse Tissues
(4 months)
*
GA
G L
evels
(µg G
AG
/mg p
rote
in)
ZFNs and hIDS transgene donor both delivered via AAV results in high levels of IDS protein secretion from the liver and significant GAG reduction in MPS II mice Mouse Plasma
Days post-injection
14
ZFN+Donor vs. MPS II Untreated
P-values: *P < 0.01; #P < 0.05
**
**
#* #
**
* * * * #
** * * * *
***
*
**
# *
Systemic delivery of ZFN mRNA and transgene donor AAV vectors may allow for in vivo correction of monogenic disease
15
AAV Vector
ZFN1
ZFN2
LNP: ZFN mRNA
Homology HomologyhIDS
One-timeperipheral IV
administration of donor vector
Repeat administrationof ZFN mRNA
Packaging DeliveryProgressive
Targeted Gene Insertion
1 2 3 4
Targ
ete
d I
nsert
ion
(%
)
Pro
tein
Exp
ressio
n
Doses
Therapeutic
Level
Targets IncludeIDS, IDUA, FIX
Repeat dosing of ZFN mRNA-LNP with a single dose of hIDS donor AAV leads to increasing levels of IDS enzymatic activity in mice
16
Pla
sm
a
IDS
En
zym
ati
c A
cti
vit
y
(nm
ol/
hr/
mL
)~2 to 3-Fold
Improvement Per Dose
0
500
1000
1500
1717
TG H
Oligo
Targeted Integration
Gene Correction
Gene Knockout
Repression
ActivationGen
e
Reg
ula
tio
n
H
Ge
no
me
Ed
itin
g
Versatile Delivery
Tissue-Specific
Viral & Non-Viral
Ex Vivo
In Vivo
Liver CNS Lung EpitheliumHem. B, MPS I & II Tau, C9ORF72 CF
T / NK Cells HSCs Airway SCsOncology, HIV β-Thal/SCA, HIV CF
Gen
e
Th
era
py
Gene Addition
ZFNs
Sangamo Technology Platform
14 Days
Lung
Liver
Collect Organs
Experimental Design: Evaluating lung-targeted LNP in mice
18
mRNAZFNs targeting CFTR intron 1
RouteTail vein i.v. (200 uL, 1 injection)
Delivery reagentInvivofectamine® Lung (ThermoFisher Scientific)
mRNA-LNP Delivery Single Cell
Dissociation and Sorting
Analyze Genome Editing
Epithelial Cells (CD326)
Bulk Tissue
>10% genome editing of lung epithelial cells following i.v. delivery of ZFN mRNA-LNP in wildtype mice
19
Lung epithelial cells
Editing occurs preferentially in lung compared to liver
20
Lung epithelial cells
Bulk liver tissue
LNP delivery enables efficient ZFN-mediated genome editing of multiple therapeutic gene targets
21
mRNA-LNP delivery of ZFNs targeting TTR and PCSK9 results in >90% circulating protein knockdown in wildtype mice
Repeat dosing of ZFN mRNA-LNP with a single human IDS AAV donor dose enables progressively increasing enzymatic activity levels in mouse plasma
mRNA-LNP delivery of ZFNs targeting CFTR achieves >10% genome modification in wildtype mice lung epithelial cells following a single dose
Ongoing work in non-viral in vivo genome editing field: Achieve therapeutically relevant levels of genome modification with an acceptable toxicity profile in non-human primate models
Research
Matt Mendel
Ken Kim
John Jascur
Gary Lee
Mike Holmes
Russ DeKelver
Development
Kathy Meyer
Carolyn Gaspar
Lisa King
Technology
Ed Rebar
Lei Zhang
Dave Paschon
Jeff Miller
Miseq Team
Production Team
Acuitas Therapeutics
Barb Mui
Ying Tam
Paulo Lin
Chris Barbosa
Tom Redelmeier
Tom Madden
Clinical
Ed Conner
Project Management
Rainier Amora
Acknowledgements
Tech Ops
AAV TeamExecutive
Sandy Macrae
Thermo Fisher Scientific
Shikha Mishra
Xavier de Mollerat du Jeu
Intellectual Property
Susan Abrahamson