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Treating Lupus: SLE Pathogenesis and Targeted Therapies
Laurence Menard, Translational Immunologist, Bristol-Myers Squibb
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Thursday, November 28, 2017
World AIDS Day and the Fight Against HIV: Discovering and Developing Emtricitabine
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16/11/2017
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“Fighting Sickle Cell Disease with Gene Correction Technology”
Alyson WeidmannManaging Editor, ACS Chemical Biology,
ACS Chemical Neuroscience, and
Biochemistry
Mark DeWittProject Scientist, Innovative
Genomics Institute, UC Berkeley
12
Fighting Sickle Cell Disease with
Gene Correction Technology
Mark DeWitt, PhDProject Manager, Innovative Genomics Institute
16/11/2017
7
Sickle Cell: Complex Disease, Simple Cause
13
Inherited recessive disease, caused by a SNP in ß-globin (HBB)
• Sickle red blood cells clog blood vessels, causing acute pain “crises” and vasculopathy
• Organ damage/organ failure
• Increased risk of stroke, pulmonary hypertension, and ACS
• 25-30 year decrement in lifespan (US)
100,000 affected in the US (almost all African-American), millions more worldwide (mostly sub-Saharan Africa)
Gene Correction of the SCD SNP
14
HBB
SNP
SCD
Healthy
• Clinical Precedent
• Sequence replacement via homology-directed repair (HDR)
16/11/2017
8
Cas9 ribonucleoprotein (RNP)/ssODN co-delivery
15
• Fast
• Easily designed
• Easily optimized
• Adaptable
• Modular
What is the biggest technical limitation to gene correction of hematopoietic stem cells in the clinic?
16
• Poor delivery of the site-directed nuclease
• Corrected alleles are not maintained long-term
• Toxicity of the gene editing procedure
• There are no technical limitations to date
Challenge Question
ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
16/11/2017
9
How Much Correction? HCT Donor Chimerism
17
Months Post BMT
20
40
60
80 • Donor chimeras indicate that low
levels of correction can cure SCD
• Possible that as little as 3-10% cellular
correction is required for event-free
survival
• “Low bar” for long-term engraftment
Walters MC, et al, Biology of Blood and Bone Marrow Transplantation (2001), 7:665-673
Gene Correction Strategy
18
• Cas9 RNP loaded with “G10” guide
• ssDNA HDR donor “CJ6” asymmetrically designed around the cut site
• co-delivery by electroporation
Richardson CD et al. NBT (2015) DeWitt MA et al. Sci. Tran. Med. (2016)
TCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC
ATGGTGCACCTGACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGG
PAM
SCD
SNP
WT
ssDNA DONOR (TOP STRAND)
CUT
SITE
AA G
16/11/2017
10
Edited SCD HSPCs show increased HbA, HbF
19
Template
pmol trG10 RNP 100 100 200
T88-107S T111-57S T111-27S-
-
GENOTYPE PHENOTYPE
T88-107S T111-57S T111-27STemplate
pmol trG10 RNP 100 100 200
-
-
0
50
100
%h
em
og
llb
in b
y H
PL
C
HbF
HbA
HbS
HbA2
Untreated 200 pmol RNP
T111-27SHbS
HbS
HbA
HbA
HbA2
HbA2
HbF
HbF
Engraftment of Edited Cells
20
Wee
k 5
Blood
Wee
k 8
Blood
Wee
k 16
(BM
)0
20
40
60
80
hu
ma
n C
D4
5+
(% o
f to
tal C
D4
5+
) Uninjected
Injected
Input
0
20
40
60
80
100
%A
llele
s b
y N
GS
%HDR
%Indel
%Total
Wee
k 8
(blood
)
Wee
k 16
(splee
n)
Wee
k 16
(BM
)0
2
4
6
8
%H
DR
by N
GS
Wee
k 8
(blood
)
Wee
k 16
(splee
n)
Wee
k 16
(BM
)0
20
40
60
80
%In
de
l by N
GS
Wee
k 8
(blood
)
Wee
k 16
(splee
n)
Wee
k 16
(BM
)0
2
4
6
8
%H
DR
by N
GS
Wee
k 8
(blood
)
Wee
k 16
(splee
n)
Wee
k 16
(BM
)0
20
40
60
80
%In
de
l by N
GS
Wendy Magis, David Martin
16/11/2017
11
PRECLINICAL DEVELOPMENT
21
Preclinical Development Timeline
22
STAGE TASKS
Target Identification -Sickle mutation
-HSPCs
-Cas9 RNP
Preclinical Proof of Concept pre-pre-IND meeting -Protocol optimization
-Validation in mouse
-Clinical scale-up
Safety Studies-Off-target qualification
-Cas9 retention
-Expansion, viability
-CFU
Draft Clinical Protocol IND
-Plerixaflor mobilization
-15 month FU
-12 patients, single-dose
-GLP NGS, immune response
pre-IND meetingGLP Test Product Manufacture
GLP Toxicity Studies
-Tox: Clinical-scale transplant
-Activity: NGS at 4 months
16/11/2017
12
CIRM TRAN1 Disease Team
23
• Mark Walters (CHO/UCSF) – SCD BMT specialist (Project PI)
• Jacob Corn (IGI/Berkeley) – CRISPR/Cas9
• Donald Kohn (UCLA) – Gene editing and gene therapy
• David Martin (CHORI/UCSF) – Globin genetics
• Project Manager – Yours Truly
Genomic
DNA
QC
Manifest
NGS Prep
(HBB and OTs)
MiSeq
sequencingAnalysis pipeline
JON
STACIA
Data!
24
High-Throughput NGS platform Jon Vu, Nick Bray, Stacia Wyman
16/11/2017
13
Optimization of Editing Conditions
25
• EP pulse: Lonza 4d
(ER100)
• sgRNA (3xMS protection)
• Culture time
before/after EP, LT-HSC
expansion additives
1 - E
R10
0
2 - D
O10
0
3 - E
O10
0
4 - C
A13
7
5 - u
ntre
ated
0
20
40
60
Condition
%A
llele
s b
y N
GS
%NHEJ
%HDR
1 - E
R10
0
2 - 3
xMS
3 - 1
xMSP
4 - u
ntre
ated
0
20
40
60
sgRNA
%A
llele
s b
y N
GS
%NHEJ
%HDR
Titration of RNP and ssDNA
26
0 50 100 150 2000
20
40
60
80
100
RNP dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
RNP titration: editing outcomes
%NHEJ
%total editing
0 50 100 150 2000
5
10
15
20
25
RNP dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
RNP titration: HDR
0 50 100 150 20040
50
60
70
80
90
ssODN dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
ssODN titration: editing outcomes
%NHEJ
%total editing
0 50 100 150 2000
5
10
15
20
25
ssODN titration: HDR
ssODN dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
0 50 100 150 2000
20
40
60
80
100
RNP dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
RNP titration: editing outcomes
%NHEJ
%total editing
0 50 100 150 2000
5
10
15
20
25
RNP dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
RNP titration: HDR
0 50 100 150 20040
50
60
70
80
90
ssODN dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
ssODN titration: editing outcomes
%NHEJ
%total editing
0 50 100 150 2000
5
10
15
20
25
ssODN titration: HDR
ssODN dose (pmol/200,000 cells)
%A
llele
s b
y N
GS
Dosing: 75 pmol RNP
per 200,000 cells, 100
pmol ssDNA per
200,000 cells
20 µM EP volume.
Dosing, cells and EP
volume will be scaled
up accordingly.
16/11/2017
14
Tranfusion Discard HSPC
27
• From transfusion of discard material of individuals with SCD undergoing treatment at Children’s
Hospital Oakland
• Discard sent to Allcells for RBC depletion and CD34 isolation
• 9 million CD34+ HSPC from ~1 liter of discard
mob
ilize
d PBSC
Sickle
PBSC
mob
ilize
d PBSC (e
dite
d)
Sickle
PBSC (e
dite
d)0
10
20
30
40
50
editing outcomes
Sample
%A
llele
s b
y N
GS
%NHEJ
%HDR
mob
ilize
d PBSC
Sickle
PBSC
mob
ilize
d PBSC (e
dite
d)
Sickle
PBSC (e
dite
d)0
2
4
6
4050607080
Condition
%C
ells
FACS analysis - stringent gating
LT-HSC (%)
ST-HSC (%)
progenitors (%)
Jenny Shin, Wendy Magis, David MartinNo colonies in CFU Assay
Mobilized PB HSPC
28
• From a mobilization of a participant in an SCD gene therapy clinical trial
• We received 130 million cells from the discard of the first collection
SCD H
SPC s1
SCD H
SPC s2
Une
dite
d0
20
40
60
80
NGS data
%A
llele
s b
y N
GS
%HDR (Correction)
%NHEJ
• Perform identically to WT-HSPC in all key respects
• Additional are possible Q3 this year, Q1 next
Form colonies in CFU assay Jenny Shin, Wendy Magis, David Martin
16/11/2017
15
HBB-s
cd_n
o-inde
l
HBB-w
t
HBD
HBG1
HBG
20
10
20
30
40
50
ß-globin expression by RNAseq (liquid culture)
%of ß
-glo
bin
tra
nscripts
(R
NA
seq)
In vitro phenotyping: HPLC and RNAseq
29Wendy Magis, David Martin
HPLC of edited P-only SCD HSPC
after correction with ssDNA donor:
22% HbS, 40% HbA, 37% HbF
RNAseq: >50% non-sickle HBB
Bringing It All Together: Ongoing Mouse Studies
30
Completion: December 2017
16/11/2017
16
SAFETY AND TOXICITY
31
In terms of safety and toxicity of ex vivo gene editing of hematopoietic stem cells with Cas9 ribonucleoprotein-based treatment, what is the foremost safety concern?
32
• Cytotoxicity of gene editing treatment
• Pre-existing immunity against Cas9 protein
• Toxicity of myeloablative regimen
• Off-target genotoxicity
Challenge Question
ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
16/11/2017
17
Pre-clinical Off-target Evaluation
33
Off-target Discovery
Validation by deep
sequencing of test product
Unbiased: GUIDE-seq, IDLV integrationBioinformatic (Cas-OFFfinder, CRISPor)
Validation in target cell type is crucial!
GUIDE-seq Identifies Few off-Targets
34
1 2310 20
C C G T T A C T G C C C T G T G G G G C A A G Reads
• • • • • • • • • • • • • • • • • • • • • • • 10776 HBBOT1 (Intergenic)• • C • • • • • • • • • • • • • • • • • T G A 2960
HBBOT1 (Intergenic)Intergenic Chr12
Intergenic Chr17Intergenic Chr17
1 2310 20
C C G T T A C T G C C C T G T G G G G C A A G Reads
• • • • • • • • • • • • • • • • • • • • • • • 295962• • C • • • • • • • • • • • • • • • • • T G A 114024• • T • • G • • • • • • • • • • • • • • • G C 5350• • C • • • • • • • • • • • • C T • • G G C A 672• • T • • • • • • • • • • • • • • • A G • G • 35
K562 Cells
HSPCs
Tsai SQ et. al. Nature Biotechnology (2015)
Shirley Shao
16/11/2017
18
Validation of GUIDE-seq Sites by Amplicon Re-sequencing
35
On-
targ
et (H
BB)
Site
1
Site
2
Site
3
Site
4
Site
5
Site
6
Site
7
Site
8
Site
9
Site
10
Site
11
Site
12
Site
13
Site
14
Site
15
Site
16
Site
17
Site
18
Site
19
Site
20
Site
21
Site
22
Site
23
(OT2)
Site
24
Site
25
Site
26
(OT1)
0.001
0.01
0.1
1
10
100
GUIDE-seq site
%In
dels
by N
GS
K562 - J10
HSPCs - J10
K562 - Un
HSPCs - Un
Shirley Shao
High-fidelity Cas9 Variants
36
IDT m
utan
t 1
IDT m
utan
t 2
espC
as91
.1
WT (B
erke
ley)
untre
ated
IDT m
utan
t 1
IDT m
utan
t 2
espC
as91
.1
WT
(Ber
keley)
untre
ated
IDT m
utan
t 1
IDT m
utan
t 2
espC
as91
.1
WT
(Ber
kele
y)
untre
ated
0.0
0.2
0.4
1.0
1.5
2.0
2.5
20
40
60
80
Cas9 variant
%M
odifi
catio
n b
y N
GS
Cutting by Cas9 variants: standard RNP assembly protocol
HBB
OT1
OT2
• IDT mutant 1 can be substituted for WT (20x reduced off-target)
• Available commercially very soon.
IDT
mut
ant 1
IDT
mut
ant 2
WT (B
erke
ley)
espC
as91
.1
untre
ated
0
20
40
60
80
100
Cas9 variant
%V
iable
(tr
ypan b
lue)
High-Fidelity Cas9s - viability 24 h after EP
(standard assembly protocol)
IDT variant (Alt-R Cas9) delivers
20x improved fidelity
16/11/2017
19
Translocations by ddPCR
37K56
2-wild
-type
K56
2-ID
T HiF
I
HSPC-ID
T HiF
i
K56
2-Une
dite
d
HSPC-U
nedite
d
K56
2-wild
-type
K56
2-ID
T HiF
I
HSPC-ID
T HiF
i
K56
2-Une
dite
d
HSPC-U
nedite
d0.00
0.05
0.10
0.15
%ddP
CR
events
(norm
aliz
ed)
OT1R
OT1F
Zule Romero (Kohn Group)
Off-target Deliverables
38
• Evaluate GUIDE-seq hits and top 200 bioinformatic hits by deep sequencing
• ddPCR (and AMPseq) for translocations between HBB and other sites such as OT1
16/11/2017
20
Cas9 Retention: Western Blot
39
100kDa
100kDa
150kDa
150kDa
37kDa
50kDa
Ladd
er
UNTR
EATED -T
ryp
UNTR
EATED +
Tryp
EP -Try
p Day
0
EP -Try
p Day
1
EP -Try
p Day
4
EP -Try
p Day
6
EP +Try
p Day
0
EP +Try
p Day
1
EP +Try
p Day
4
EP +Try
p Day
6
20ng10ng
5ng4ng
2ng1ng
0.5ng
0.1ng
0ng
K562 Cells
Cas9
GAPDH
100kDa
150kDa
37kDa
50kDa
UNTR
EATED -T
ryp
EP -Try
p Day
1
EP +Try
p Day
1
Ladd
er
HSPCs
Cas9 apparent by blot 1 day after EP
Likely on the surface of the cells
Jon Vu
y=0.057x+0.0093R²=0.95839
y=0.058x+0.1101R²=0.94778
y=0.0685x+0.0209R²=0.96254
0
0.05
0.1
0.15
0.2
0.25
0.3
0 0.5 1 1.5 2 2.5
Absorbance450-655nm
ngCas9StandardAdded
Cas9reten oninHSPCs1daya erEP
Untreated(-trypsin)
EP(day1)-trypsin
EP(day1)+trypsin
Linear(Untreated(-trypsin))
Linear(EP(day1)-trypsin)
Linear(EP(day1)+trypsin)
Cas9 Retention: ELISA
40
Jon Vu
• ~15 µg Cas9
protein per
Clinical
equivalent
• +Trypsin leads
to 10-fold
reduction
• Most Cas9 is
on cell
surface
16/11/2017
21
MANUFACTURING (CMC)
41
For pre-clinical studies, what grade of reagents must be used to manufacture a test product?
42
• Reagents made in your lab (not under GLP)
• Research-grade reagents from a qualified supplier
• Reagents made under cGMP
• All of the above
Challenge Question
ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
16/11/2017
22
Preclinical Supplier Qualification Program
43
Critical Reagent? Low Risk Supplier?
In-House Testing req.?
No
No
Yes
Yes
Yes
Pass
Pass
Accept on CoA
Acceptance with testing
In-house testing
Desk Audit
Site VisitFormulation and Release
Potency: %HDR
Safety: Viability, expansion
Purity: on CoA
cGMP?
FDA-audited?
ISO cert. QMS?
Track record?
Questionnaire
Doc. Review
Phone Call(s)
Interviews
Doc. Review
Concentration, solvent
Release Testing
• “Phase-appropriate” raw materials program
• Test manufacture and GLP toxicity studies using research-grade reagents
• Phase I product using cGMP reagents where possible/appropriate
YES
YES
NO
NO
YES
PASS
PASS
Manufacturing at UCLA
44
Release:
Potency (%Correction)Cas9 retained (ELISA)Identity (%CD34)Viability (trypan blue)Sterility, MycoplasmaEndotoxin
16/11/2017
23
CLINICAL
45
Study Schema / Methodology
46
Enrollment Critical Reagents: Cas9, ssDNA, sgRNA
Clincial: UCLA, CHO (UCSF) Manufacturing: UCLA
Gene Correction (EP)
Cryopreservation
Release Testing
B-L measurement
HSPC Mobilization
CD34+ Isolation
HSPC backup
Myeloablation
HSPC infusion
Follow-up (24 mo)
LTFU (15 yr)
Baseline evaluation/eligibility: physical, laboratory exams, history
Mobilization: single-dose plerixafor. 1.5 milion cells/kg for manufacturing,
~0.5 million/kg backup.
Myeloablation: busulfan
Follow-up: monthly blood draws.
LTFU: every 6 months, in-person, 15 years.
Enrollment Critical Reagents: Cas9, ssDNA, sgRNA
Clincial: UCLA, CHO (UCSF) Manufacturing: UCLA
Gene Correction (EP)
Cryopreservation
Release Testing
B-L measurement
HSPC Mobilization
CD34+ Isolation
HSPC backup
Myeloablation
HSPC infusion
Follow-up (24 mo)
LTFU (15 yr)
Enrollment Critical Reagents: Cas9, ssDNA, sgRNA
Clincial: UCLA, CHO (UCSF) Manufacturing: UCLA
Gene Correction (EP)
Cryopreservation
Release Testing
B-L measurement
HSPC Mobilization
CD34+ Isolation
HSPC backup
Myeloablation
HSPC infusion
Follow-up (24 mo)
LTFU (15 yr)
16/11/2017
24
Correlative Study: Ineffective Erythropoiesis
47
Earlier studies suggest ineffective
erythropoiesis in BM of SCD patients
with WT donor chimerism.
This study generates 3 major
genotypes: SCD, thalassemia-like,
and wild-type (corrected). Which
one(s) is predominant in BMMCs,
PBMCs, and RBCs?
LessMature
MoreMature
Wu et al. (2005) Blood
Expected Results / Potential Problems
48
Optimal outcomes include:
• Replacement of HbS by HbA and HbF following DP infusion.
• Elimination of all signs and symptoms of sickle cell anemia with a significant
survival benefit
Minimally acceptable outcomes include:
• Reduction of HbS (<50%) in the absence of RBC transfusion support
• Other biological improvements in RBC physiology, such as RBC survival
Potential problems
• Clonal expansion resulting from off-target mutagenesis
• Immune response resulting from residual Cas9 protein in the DP
16/11/2017
25
Discover More…
49
https://innovativegenomics.org
Acknowledgements
50
THE SCD TEAM
Wendy Magis (CHORI)
Nicolas Bray (IGI)
Stacia Wyman (IGI)
Jenny Shin (IGI)
Jonathan Vu (IGI)
Jacob Corn (IGI)
David Martin (CHORI)
Mark Walters (CHO)
Don Kohn (UCLA)
Zulema Garcia Romero (UCLA)
Anastasia Lemova (UCLA)
Seok-Jin Heo (CHORI)
Dario Boffelli (CHORI)
Jennifer Chung (IGI)
FUNDINGCIRM TRAN1-09292
CIRM CESCG
IGI-Li Ka Shing Foundation
16/11/2017
26
The Community of Chemical Biologists
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for the rapid communication of research at the
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Follow us on Twitter:
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Submit your research today!
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Proudly co-produced with ACS Publications 52
www.acs.org/acswebinars
Slides available now! Recordings are an exclusive ACS member benefit.
“Fighting Sickle Cell Disease with Gene Correction Technology”
Alyson WeidmannManaging Editor, ACS Chemical Biology,
ACS Chemical Neuroscience, and
Biochemistry
Mark DeWittProject Scientist, Innovative
Genomics Institute, UC Berkeley
16/11/2017
27
53
Upcoming ACS Webinarswww.acs.org/acswebinars
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Thursday, November 30, 2017
Treating Lupus: SLE Pathogenesis and Targeted Therapies
Laurence Menard, Translational Immunologist, Bristol-Myers Squibb
Mary Struthers, Drug Discovery Biologist, Bristol-Myers Squibb
Thursday, November 28, 2017
World AIDS Day and the Fight Against HIV: Discovering and Developing Emtricitabine
Dennis Liotta, Associate Director, The Emory Center for AIDS Research
Nicholas Meanwell, Executive Director, Bristol-Myers Squibb
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Upcoming ACS Webinarswww.acs.org/acswebinars
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Thursday, November 30, 2017
Treating Lupus: SLE Pathogenesis and Targeted Therapies
Laurence Menard, Translational Immunologist, Bristol-Myers Squibb
Mary Struthers, Drug Discovery Biologist, Bristol-Myers Squibb
Thursday, November 28, 2017
World AIDS Day and the Fight Against HIV: Discovering and Developing Emtricitabine
Dennis Liotta, Associate Director, The Emory Center for AIDS Research
Nicholas Meanwell, Executive Director, Bristol-Myers Squibb