Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with … · 2020-07-02 · hU6 sgRNA TBG...
Transcript of Amelioration of Alpha-1 Antitrypsin Deficiency Diseases with … · 2020-07-02 · hU6 sgRNA TBG...
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