1© 2017 Editas Medicine© 2017 Editas Medicineeditasmedicine.com

Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with Genome Editing

in Transgenic MiceShen Shen1, Minerva E. Sanchez1, Erik Corcoran1, Keith

Blomenkamp2, Eugenio Marco1, Jeffrey H. Teckman2, David Bumcrot1

1Editas Medicine, Cambridge, Massachusetts, USA2Department of Pediatrics, St. Louis University School of Medicine, St.

Louis, Missouri, USAASGCT 2017

2© 2017 Editas Medicine

Disclosures

▪ S.S., M.E.S, E.C., E.M., and D.B. are employees at Editas Medicine.▪ Dr. Jeffrey H. Teckman is a consultant to Editas Medicine. Keith Blomenkamp

is employed by Saint Louis School of Medicine.

3© 2017 Editas MedicineFairbanks, KD, American Journal of Gastroenterology (2008); Lomas, DA, Clin Med (2005); PDB ID: 3NE4

Alpha-1 Antitrypsin Deficiency

PolymerizationMisfolding

Z(E342K)

AAT Protein

Liver Globules byPeriodic Acid-Schiff Staining

Fibrosis, Cirrhosis, Hepatocellular Carcinoma

Alveolar Damage

Emphysema, COPD

+

4© 2017 Editas MedicineCarlson JA, JCI, 1989; Rudnick DA, Hepatology, 2004

PiZ Transgenic Mice Recapitulate Liver Phenotypes

▪ Transgenic mice harbor the intact human SERPINA1-Z locus (PiZ)▪ Positive staining of hAAT-Z globules with Periodic Acid Schiff + Diastase (PAS-D)▪ Mouse SerpinA1 loci are still present

PiZ Mouse Wildtype

PAS-D Staining

Human SERPINA1

IA IB IC II III IV V5’UTR 3’UTR

Z Mutation: E342K

5© 2017 Editas Medicine

hSERPINA1 Gene Editing to Treat AATD Diseases

Human SERPINA1

IA IB IC II III IV V5’UTR 3’UTR

Z Mutation: E342K

HDR Strategy

II

Non-Homologous End JoiningNonsense-mediated Decay Homology-Directed DNA Repair

Reduce AAT-Z Globules

Alleviate Liver Burden

Improve Lung Protection

NHEJ Strategy

+-+ +

++

V

Z Mutation: E342K

Donor

6© 2017 Editas Medicine

Gene Editing of Exon II Decreases hSERPINA1 Expression

sgRNA TBG saCas9hU6

4.7 kb

AAV8

P B S A A V 8 -C R IS P R-1 0

0

1 0

2 0

3 0

4 0

5 0

Pe

rce

nt

Ed

itin

g

34%

NGS

P B S A A V 8 -C R IS P R0

2 0

4 0

6 0

8 0

1 0 0

hS

ER

PIN

A1

Ex

pre

ssio

n

rela

tiv

e t

o B

2m

by

RT-

qP

CR

4%

RT-qPCR

7© 2017 Editas Medicine

Gene Editing of Exon II Reduces Circulating AAT-Z

0 1 0 2 0 3 0 4 01 0 1

1 0 2

1 0 3

D a y s p o s t in je c t io n

Se

rum

hA

AT

(P

g/m

l)

P B SA A V 8 -C R IS P R

22%

1%

ELISA of Human AAT in Mouse Serum

8© 2017 Editas Medicine

Gene Editing of Exon II Reduces AAT-Z Globules in Liver

P B S A A V 8 -C R IS P R0

1u1 0 5

2u1 0 5

3u1 0 5

To

tal

are

a o

f g

lob

ule

s(p

ixe

ls/f

ram

e)

12%

PAS-D Staining of Livers on Day 35

PBS

100 μm

AAV8-CRISPR

100 μm

Quantitation of PAS-D staining

9© 2017 Editas Medicine

Dual-Vector HDR Approach to Correct the Z Mutation

Human SERPINA1

IA IB IC II III IV V5’UTR 3’UTR

Z Mutation: E342K

Outcomes on Exon V

TBG saCas9

AAV8

Donor

1.28e14 vg/kg

Cas9

5.0e13 vg/kg

sgRNAhU6

Homology Arm

Homology Arm

Unedited

Insertions

Deletions

Correction by HDR

10© 2017 Editas Medicine

Efficient Reduction of hSERPINA1 Expression in vivo

0 .0

0 .5

1 .0

hS

ER

PIN

A1

ex

pre

ss

ion

rela

tiv

e t

o B

2M

PBS AAV8-Cas9+AAV8-Donor

14%

0 1 0 2 0 3 0 4 0 5 0 6 01 0 0

1 0 1

1 0 2

1 0 3

1 0 4

D a y s p o s t in je c t io n

Se

rum

AA

T (P

g/m

l)

P B S

A A V 8 -C a s 9 + A A V 8 -D o n o r

3%

1%

RNAseq of Total RNA ELISA of Human AAT in Mouse Serum

11© 2017 Editas Medicine

Wild-Type hAAT Expression Restored in PiZ Livers

0

2

4

6

8P B SA A V 8 -C a s 9 + A A V 8 -D o n o r

PBS AAV8-Cas9+AAV8-Donor

Per

cent

Cor

rect

ion

of th

e Z

Mut

atio

n

RNAseq of Total Liver RNA

5%

12© 2017 Editas Medicine

Summary

▪ NHEJ approach disrupts hSERPINA1 loci in PiZ transgenic mice, dramatically reducing AAT-Z in circulation and AAT-Z aggregation in hepatocytes

▪ HDR approach corrects the Z mutation in hSERPINA1 in vivo resulting in reduction of circulating AAT-Z and restoration of wild-type PiM expression

▪ Due to limitations in current models, a novel PiZ transgenic mouse would be required to assess the potential impact of gene correction on lung disease caused by PiZ mutations

▪ CRISRP/Cas9, in combination with AAV delivery systems, has the potential to be developed as a therapy for AATD patients with the PiZZ genotype

13© 2017 Editas Medicine

Acknowledgements

Charlie AlbrightVic MyerAndrew HackHaiyan JiangMichael StefanidakisGeorgia GiannoukosDawn Ciulla

Viral Vector CoreGuangping GaoQin SuRan HeJun Xie