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16/11/2017
“Fighting Sickle Cell Disease with Gene Correction Technology”
Mark DeWitt
Alyson Weidmann
Project Scientist, Innovative Genomics Institute, UC Berkeley
Managing Editor, ACS Chemical Biology, ACS Chemical Neuroscience, and Biochemistry
Slides available now! Recordings are an exclusive ACS member benefit.
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11
Fighting Sickle Cell Disease with Gene Correction Technology
Mark DeWitt, PhD Project Manager, Innovative Genomics Institute 12
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16/11/2017
Sickle Cell: Complex Disease, Simple Cause 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 AfricanAmerican), millions more worldwide (mostly sub-Saharan Africa) 13
Gene Correction of the SCD SNP SNP HBB
SCD
Healthy • •
Clinical Precedent Sequence replacement via homology-directed repair (HDR)
14
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16/11/2017
Cas9 ribonucleoprotein (RNP)/ssODN co-delivery
•
Fast
•
Easily designed
•
Easily optimized
•
Adaptable
•
Modular
15
Challenge Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
What is the biggest technical limitation to gene correction of hematopoietic stem cells in the clinic? • 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
16
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How Much Correction? HCT Donor Chimerism
• Donor chimeras indicate that low levels of correction can cure SCD
80 60
• Possible that as little as 3-10% cellular correction is required for event-free survival
40 20
• “Low bar” for long-term engraftment
Months Post BMT
Walters MC, et al, Biology of Blood and Bone Marrow Transplantation (2001), 7:665-673
17
Gene Correction Strategy • Cas9 RNP loaded with “G10” guide • ssDNA HDR donor “CJ6” asymmetrically designed around the cut site • co-delivery by electroporation
TCAGGGCAGAGCCATCTATTGCTTACATTTGCTTCTGACACAACTGTGTTCACTAGCAACCTCAAACAGACACC ssDNA DONOR (TOP STRAND)
SCD SNP
CUT SITE PAM
ATGGTGCACCTGACTCCTGTGGAGAAGTCTGCCGTTACTGCCCTGTGGGGCAAGGTGAACGTGGATGAAGTTGG AA G WT
Richardson CD et al. NBT (2015) DeWitt MA et al. Sci. Tran. Med. (2016)
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Edited SCD HSPCs show increased HbA, HbF Untreated
200 pmol RNP T111-27S
HbS
HbA HbA2 HbS HbF HbA2 HbF HbA
%hemogllbin by HPLC
100
HbA2 HbS HbA HbF
50
0
Template pmol trG10 RNP -
T88-107S T111-57S T111-27S
100
100
Template pmol trG10 RNP
200
-
T88-107S T111-57S
100
100
T111-27S
200
PHENOTYPE
GENOTYPE
19
Engraftment of Edited Cells 80
%HDR %Indel %Total
80
human CD45+ (% of total CD45+)
60 40 20
Injected Uninjected
60 40 20 0
In
pu
t
80
6
6
60
60
0
0
W ee k
8 W ee k
k ee W
8
d oo Bl W
k ee
16
M (B
)
40
20
20
0
0 (B M )
2
16 W (s ee ple k en 8 (b ) lo od W ) e W ek ee 16 k 16 (BM (s ) pl ee n)
2
40
d oo Bl
W ee k 16 W (s ee ple k 8 en) (b lo od W ) W eek ee 1 6 k 16 (BM (s pl ) ee n)
4
W ee k 16 W (B ee M k ) 8 (b lo od )
4
%Indel by NGS
80
%Indel by NGS
8
%HDR by NGS
8
(b lo od )
%HDR by NGS
k ee W
5
16
0
W ee k
%Alleles by NGS
100
Wendy Magis, David Martin
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PRECLINICAL DEVELOPMENT
21
Preclinical Development Timeline STAGE
TASKS -Sickle mutation
Target Identification
-HSPCs -Cas9 RNP
Preclinical Proof of Concept
pre-pre-IND meeting
-Protocol optimization -Validation in mouse -Clinical scale-up -Off-target qualification -Cas9 retention
Safety Studies
-Expansion, viability -CFU
GLP Test Product Manufacture
pre-IND meeting -Tox: Clinical-scale transplant -Activity: NGS at 4 months
GLP Toxicity Studies
-Plerixaflor mobilization
Draft Clinical Protocol
IND
-15 month FU -12 patients, single-dose -GLP NGS, immune response 22
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CIRM TRAN1 Disease Team • 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
23
High-Throughput NGS platform
Jon Vu, Nick Bray, Stacia Wyman
STACIA
Analysis pipeline
Data!
MiSeq sequencing
Manifest
Genomic DNA
JON
NGS Prep (HBB and OTs) QC
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Optimization of Editing Conditions %NHEJ
%Alleles by NGS
60
• EP pulse: Lonza 4d (ER100)
%HDR
40
20
0
• sgRNA (3xMS protection)
1
-E
R
0 10 2
-D
O
0 10 3
-E
0 10 O 4
A1 -C
37 5
nt -u
ed at re
Condition %NHEJ
• Culture time before/after EP, LT-HSC expansion additives
%Alleles by NGS
60
%HDR
40
20
0
1
-E
R
0 10 2
-3
xM
S 3
-1
xM
SP 4
at re nt -u
ed
sgRNA
25
Titration of RNP and ssDNA RNP titration: editing outcomes
ssODN titration: editing outcomes 90
%NHEJ
60
%total editing 60
20 40 0 20 0
50
0 0
100
150
%Alleles by NGS
40
90
%NHEJ
80
80 70 60 50
80
%total editing 70
%NHEJ %total editing
60 50
0 0
50
RNP titration: HDR
1515 1010 5 0
15 10 5 0
100
ssODN dose (pmol/200,000 cells) 100 150 200
25
%Alleles by NGS
20
%Alleles by NGS
25
%Alleles by NGS
%Alleles by NGS
25
50
200
ssODN titration: HDR
2020
0
150
ssODN titration: HDR
RNP titration: HDR
0
100
ssODN dose (pmol/200,000 cells)
25
0
50
40
RNP 50 dose (pmol/200,000 cells) 200 100 150
Dosing: 75 pmol RNP per 200,000 cells, 100 pmol ssDNA per 200,000 cells 20 µM EP volume.
40
200
RNP dose (pmol/200,000 cells)
5
%NHEJ
ssODN titration: editing outcomes
%total editing
100
%Alleles by NGS
RNP titration: editing outcomes
80
%Alleles by NGS
%Alleles by NGS
100
150
50dose (pmol/200,000 100 150 RNP cells)
RNP dose (pmol/200,000 cells)
200
0
200
Dosing, cells and EP volume will be scaled up accordingly.
20 15 10 5 0 50 100 150 0 100cells) ssODN dose 50 (pmol/200,000
200
150
200
ssODN dose (pmol/200,000 cells) 26
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16/11/2017
Tranfusion Discard HSPC • 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
editing outcomes 50
%Cells
%Alleles by NGS
FACS analysis - stringent gating 80 70 60 50 40
LT-HSC (%) ST-HSC (%) progenitors (%)
6 4
%NHEJ
40
%HDR
30 20 10
2
0
0
m
ob
e iliz
d
S PB
C ck Si
le
m
PB
SC
iliz ob
ed
PB
SC
di (e
t
) ed
ck Si
le
SC PB
d (e
d) ite
m
i ob
e liz
d
PB
SC Si
le ck
m
S PB
i ob
liz
C
ed
PB
SC
d) te di (e
Si
le ck
S PB
C
d) te di (e
Sample
Condition
No colonies in CFU Assay
Jenny Shin, Wendy Magis, David Martin
27
Mobilized PB HSPC • 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
NGS data
%Alleles by NGS
80
%HDR (Correction)
•
Perform identically to WT-HSPC in all key respects
•
Additional are possible Q3 this year, Q1 next
%NHEJ
60 40 20
Form colonies in CFU assay
ne di te d U
s2 C SP H D SC
SC
D
H
SP
C
s1
0
Jenny Shin, Wendy Magis, David Martin
28
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In vitro phenotyping: HPLC and RNAseq HPLC of edited P-only SCD HSPC after correction with ssDNA donor: 22% HbS, 40% HbA, 37% HbF
%of ß-globin transcripts (RNAseq)
ß-globin expression by RNAseq (liquid culture) 50 40 30 20 10
RNAseq: >50% non-sickle HBB H
BB
0
-s
l de in on _ cd
H
-w BB
t
BD H
BG H
1
BG H
2
Wendy Magis, David Martin
29
Bringing It All Together: Ongoing Mouse Studies
Completion: December 2017 30
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SAFETY AND TOXICITY
31
Challenge Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
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? • Cytotoxicity of gene editing treatment • Pre-existing immunity against Cas9 protein • Toxicity of myeloablative regimen • Off-target genotoxicity
32
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Pre-clinical Off-target Evaluation
Unbiased: GUIDE-seq, IDLV integration Bioinformatic (Cas-OFFfinder, CRISPor)
Off-target Discovery
Validation by deep sequencing of test product
Validation in target cell type is crucial!
33
GUIDE-seq Identifies Few off-Targets 1
K562 Cells
10
23
Reads
• • • • • • • • • • • • • • • • • • • • • • •
295962 114024 5350 672 35
• • • • • • • •
C T C T
1
HSPCs
20
CCGTTACTGCCCTGTGGGGCAAG
TGA • • • G C • G G C A AG • G •
• • • • • • • • • • • • • • • • • • •
G
• • • • • • • • • • • •
• • • • • • • • • • • •
CT
•
• • • • • • • • • • • • • • •
10
20
23
HBB OT1 (Intergenic) Intergenic Chr12 Intergenic Chr17 Intergenic Chr17
CCGTTACTGCCCTGTGGGGCAAG
Reads
• • • • • • • • • • • • • • • • • • • • • • •
10776 HBB 2960 OT1 (Intergenic)
• •
C
• • • • • • • • • • • • • • • • •
TGA
Shirley Shao
Tsai SQ et. al. Nature Biotechnology (2015)
34
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Validation of GUIDE-seq Sites by Amplicon Re-sequencing 100
K562 - J10 HSPCs - J10
10
K562 - Un
%Indels by NGS
HSPCs - Un 1
0.1
0.01
O
nta
rg et (H
BB Si ) te Si 1 te Si 2 te Si 3 te Si 4 te Si 5 te Si 6 te Si 7 te Si 8 t Si e 9 te Si 10 te Si 11 te Si 12 te Si 13 te Si 14 te Si 15 te Si 16 te Si 17 te Si 18 te Si 19 te Si 20 t Si S e 2 te i t 1 23 e 2 (O 2 T Si 2) t Si S e 24 te i t 26 e 2 (O 5 T1 )
0.001
GUIDE-seq site
Shirley Shao 35
High-fidelity Cas9 Variants Cutting by Cas9 variants: standard RNP assembly protocol 80
100
OT1 OT2
40 20 2.5 2.0 1.5
%Viable (trypan blue)
HBB
60
80 60 40 20
d at e
91 . un
tre
as
W
T
pC
(B
m T
es
er k
el e
an t
y)
2
1 ut
ut
0.2
ID
ID
T
m
0.4
1
0
1.0
an t
%Modification by NGS
High-Fidelity Cas9s - viability 24 h after EP (standard assembly protocol)
Cas9 variant
IDT variant (Alt-R Cas9) delivers 20x improved fidelity
ID
T ID mu T ta m n es u t t 1 W pC ant T as 2 (B 9 er 1.1 k u n e le t y ID rea ) te T d ID mu T ta m nt es u t 1 W pC ant T as 2 (B 9 er 1.1 k u n e le t y ID rea ) te T d ID mu T ta m nt u es t 1 W pC ant T as 2 (B 9 er 1.1 k u n e le tre y) at ed
0.0
Cas9 variant
• IDT mutant 1 can be substituted for WT (20x reduced off-target) • Available commercially very soon.
36
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16/11/2017
OT1R OT1F 0.10
0.05
0.00
62 K5 -wi 6 ld H 2-I -typ SP D T e K5 C-I Hi 62 DT F I H -U H SP n iF C ed i -U ite ne d K5 di 62 te d w K5 i 62 ldH -I typ SP D T e K5 C-I Hi 62 DT F I H -U H SP n iF C ed i -U i t ne ed di te d
Zule Romero (Kohn Group)
0.15
K5
%ddPCR events (normalized)
Translocations by ddPCR
37
Off-target Deliverables
• 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
38
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Cas9 Retention: Western Blot p p ry ry 0 1 4 6 -T +T 0 1 4 6 y y y y D ED ay ay ay ay E D D D D D a Da D a D a T AT p p p p A p p p p ry ry ry ry ry ry ry ry e r RE R E -T -T -T -T +T +T +T +T dd T T La UN UN EP E P E P E P EP EP EP EP
dd La
er
T UN
R
D TE EA
p ry -T
EP
p ry -T
y Da
1
EP
+
yp Tr
y Da
1
150kDa 100kDa
150kDa 100kDa
Cas9
50kDa 37kDa
50kDa 37kDa
GAPDH
HSPCs 150kDa 100kDa 0 n 0 . 0. 1 n 2 n 4 n 5 n 1 0 2 0 g 1n 5n g g g g ng ng g g
Cas9 apparent by blot 1 day after EP Likely on the surface of the cells
K562 Cells Jon Vu
39
Cas9 Retention: ELISA Cas9 reten on in HSPCs 1 day a er EP
Jon Vu
0.3
• ~15 µg Cas9 protein per Clinical equivalent
0.25
Absorbance 450-655nm
y = 0.058x + 0.1101 R² = 0.94778
0.2
• +Trypsin leads to 10-fold reduction
y = 0.0685x + 0.0209 R² = 0.96254
0.15
y = 0.057x + 0.0093 R² = 0.95839
• Most Cas9 is on cell surface
0.1 Untreated (-trypsin) EP (day 1) -trypsin
0.05
EP (day 1) +trypsin Linear (Untreated (-trypsin)) Linear (EP (day 1) -trypsin) Linear (EP (day 1) +trypsin)
0 0
0.5
1 1.5 ng Cas9 Standard Added
2
2.5 40
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16/11/2017
MANUFACTURING (CMC)
41
Challenge Question ANSWER THE QUESTION ON BLUE SCREEN IN ONE MOMENT
For pre-clinical studies, what grade of reagents must be used to manufacture a test product? • Reagents made in your lab (not under GLP) • Research-grade reagents from a qualified supplier • Reagents made under cGMP • All of the above
42
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Preclinical Supplier Qualification Program Critical Reagent?
No
NO
YES Yes
In-house testing Potency: %HDR Safety: Viability, expansion Purity: on CoA
YES Yes
Accept on CoA No
YES Yes
NO
cGMP? FDA-audited? ISO cert. QMS? Track record?
Questionnaire Doc. Review Phone Call(s)
Desk Audit
PASS Pass
In-House Testing req.?
Formulation and Release Acceptance with testing
• • •
Low Risk Supplier?
PASS Pass
Concentration, solvent Release Testing
Site Visit Interviews Doc. Review
“Phase-appropriate” raw materials program Test manufacture and GLP toxicity studies using research-grade reagents Phase I product using cGMP reagents where possible/appropriate 43
Manufacturing at UCLA
Release: Potency (%Correction) Cas9 retained (ELISA) Identity (%CD34) Viability (trypan blue) Sterility, Mycoplasma Endotoxin
44
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CLINICAL
45
Study Schema / Methodology Clincial: CHO CHO (UCSF) Clincial: UCLA, (UCSF) Clincial:UCLA, UCLA, CHO (UCSF)
Manufacturing: UCLA Manufacturing: UCLA UCLA Manufacturing:
Enrollment
Critical Reagents: Cas9, ssDNA, sgRNA
Enrollment Enrollment
Critical Reagents: Cas9, ssDNA, Critical Reagents: Cas9, sgRNA ssDNA, sgRNA
B-L measurement
CD34+ Isolation
B-L measurement B-L measurement
CD34+ Isolation CD34+ Isolation Gene Correction (EP)
HSPC Mobilization
HSPC Mobilization HSPC Mobilization Myeloablation
HSPC backup
HSPC backup HSPC backup
Myeloablation Myeloablation HSPC infusion
HSPC infusion HSPC infusion Follow-up (24 mo)
Follow-up (24 mo) Follow-up LTFU (15 (24 yr) mo)
Gene Correction (EP) Gene Correction (EP) Cryopreservation
Cryopreservation Cryopreservation Release Testing
Baseline evaluation/eligibility: physical, laboratory exams, history Release Release Testing Testing 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. 46
LTFU (15LTFU yr) (15 yr)
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16/11/2017
Correlative Study: Ineffective Erythropoiesis Less Mature
More Mature
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?
Wu et al. (2005) Blood
47
Expected Results / Potential Problems 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 (
