we will begin momentarily at 2pm et · 2017-11-16 · -t88-107s t1 11-57s t1 11-27s-genotype...

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Thursday, November 30, 2017

Treating Lupus: SLE Pathogenesis and Targeted Therapies

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Proudly co-produced with ACS Publications 11


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

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

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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


%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


%A

llele

s b

y N

GS

0 50 100 150 2000

20

40

60

80

100


%A

llele

s b

y N

GS

RNP titration: editing outcomes

%NHEJ

%total editing

0 50 100 150 2000

5

10

15

20

25


%A

llele

s b

y N

GS

RNP titration: HDR

0 50 100 150 20040

50

60

70

80

90


%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


%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


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


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.




Cryopreservation

Release Testing

B-L measurement

HSPC Mobilization

CD34+ Isolation

HSPC backup

Myeloablation

HSPC infusion

Follow-up (24 mo)

LTFU (15 yr)




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

ACS Chemical Biology is an international forum

for the rapid communication of research at the

interface of chemistry and biology

Editor-in-Chief:

Laura Kiessling (MIT)

Follow us on Twitter:

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Submit your research today!

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“I found this to be very timely and relevant to our efforts to develop a more robust laboratory safety program that includes an emphasis on security. This ACS Webinar mentioned some points that I had not considered, but now I will add them to our program.”

Holly Gates-Mayer, Biosafety OfficerEnvironmental Health and SafetyUniversity of Colorado Boulder

Be a featured fan on an upcoming webinar! Write to us @ [email protected]

How has ACS Webinars benefited you?

http://bit.ly/ChemThreat

®

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