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

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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

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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

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• 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

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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

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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

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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

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>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

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ZFNs enable genome editing

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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

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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

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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

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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

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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

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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

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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

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Lung epithelial cells

Editing occurs preferentially in lung compared to liver

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Lung epithelial cells

Bulk liver tissue

LNP delivery enables efficient ZFN-mediated genome editing of multiple therapeutic gene targets

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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