crispr/cas9 technology for gene therapy and epigenome …...crispr/cas9 technology for gene therapy...

CRISPR/Cas9 Technology for

Gene Therapy and Epigenome Editing

Charles A. Gersbach, Ph.D.Department of Biomedical Engineering

Department of Orthopaedic Surgery

Center for Genomic and Computational Biology

Duke University

May 18, 2016

American Association of Pharmaceutical Scientists

National Biotechnology Conference

Boston, MA

Charles A Gersbach is a Scientific Advisor

to Editas Medicine, Inc.

This relationship is managed by Duke University.

Disclosures

Genome Engineering: The Next Phase of the Genomic Revolution

• Genome sequencing

• Genome annotation

• Genome-wide association studies

• Need tools to engineer the genome• Basic science

• Biotechnology

• Medicine

• Synthetic biology

Genome Engineering

Gene TherapyOusterout et al, Molecular Therapy (2013)

Ousterout et al, Molecular Therapy (2015)

Ousterout et al, Nature Communications (2015)

Nelson et al, Science (2016)

Epigenome EditingHilton et al, Nature Biotechnology (2015)

Polstein et al, Genome Research (2015)

Thakore et al, Nature Methods (2015)

Gene Regulation and Cell FatePerez-Pinera et al, Nature Methods (2013a)

Perez-Pinera et al, Nature Methods (2013b)

Kabadi et al, Nucleic Acids Research (2014)

Kabadi et al, ACS Syn Biol (2015)

OptogeneticsPolstein et al, JACS (2012)

Polstein et al, Nature Chemical Biology (2015)

WT mdx mdx + CRISPR

Genome Editing with Engineered Nucleases

+ Nuclease(s)

Gene Disruption

(Non-Homologous

End Joining)

Gene

Addition/Exchange

(Homologous

Recombination)

Target Gene

Gene Deletion

(Non-Homologous

End Joining)

Opportunity for precise and reproducible genetic engineering

Rouet et al., PNAS 1994: DNA breaks lead to efficient genome editing

Homologous recombination in human cells ~10-6-10-9

In the presence of DNA breaks ~10-1-10-3

Programmable Nucleases

DNA-binding domains:

• Zinc finger proteins

• TAL effectors

Effector domains:

• FokI endonuclease

catalytic domain

Zinc Finger Nucleases (ZFNs)

and TALENs

Cas9

gRNA

CRISPR/Cas9

Genome Editing with Engineered Nucleases

+ Nuclease(s)

Gene Disruption

(Non-Homologous

End Joining)

Gene

Addition/Exchange

(Homologous

Recombination)

Target Gene

Gene Deletion

(Non-Homologous

End Joining)

Correction of Genetic Diseases

by Genome Editing:

X-SCID: Urnov et al, Nature (2005)

Hemophilia: Li et al, Nature (2011)

Sickle Cell Disease: Zou et al, Blood (2011)

Sebastiano et al, Stem Cells (2011)

Alpha-1-antitrypsin: Yusa et al, Nature (2011)

HIV (Perez, E. E. et al., (2008); Holt, N. et al., (2010); Mussolino, C. et al., (2011); Wilen, C. B. et al., (2011); Li, L. et al.,

(2013); Mandal, P. K. et al., (2014); Tebas, P. et al., (2014); Ye, L. et al., (2014); Sather, B. D. et al., (2015); Badia, R.

et al., (2014); Fadel, H. J. et al., (2014); Hu, W. et al., (2014); Voit, R. A. et al., (2013))

Hepatitis B virus (Cradick, T. J. et al., (2010); Bloom, K. et al., (2013); Chen, J. et al., (2014); Lin, S. R. et al., (2014);

Weber, N. D. et al., (2014); Dong, C. et al., (2015); Kennedy, E. M. et al., (2015); Liu, X. et al., (2015))

Herpes simplex virus (Grosse, S. et al., (2011); Aubert, M. et al., (2014); Bi, Y. et al., (2014))

Human papilloma virus (Kennedy, E. M. et al., (2014))

T Cell Immunotherapy (Provasi, E. et al., (2012); Torikai, H. et al., (2012); Berdien, B. et al., (2014); Boissel, S. et al., (2014);

Torikai, H. et al., (2013); Abrahimi, P. et al., (2015); Reik, A. et al., (2007); Beane, J. D. et al.,

(2015); Schumann, K. et al., (2015))

Immunodeficiencies (Urnov, F. D. et al., (2005); Genovese, P. et al., (2014); Joglekar, A. V. et al., (2013); Rahman, S. H. et al.,

(2015))

Sickle cell disease and β-thalessemia (Sebastiano, V. et al., (2011); Xie, F. et al., (2014); Huang, X. et al., (2015); Hoban, M.

D. et al., (2015); Bauer, D. E. et al., (2013); Canver, M. C. et al., (2015); Vierstra, J. et al., (2015))

Liver-Targeted Gene Editing

Hemophilia (Li, H. et al., (2011); Anguela, X. M. et al., (2013))

Enzyme replacement(Barzel, A. et al., (2015); Sharma, R. et al., (2015))

Tyrosinemia type I (Yin, H. et al., (2014))

PCSK9 (Ding, Q. et al., (2014); Ran, F. A. et al., (2015))

α-1-antitrypsin deficiency (Yusa, K. et al., (2011))

Duchenne muscular dystrophy (Popplewell, L. et al., (2013); Li, H. L. et al., (2015); Benabdallah, B. F. et al., (2013);

Ousterout, D. G. et al., (2013); Li, H. L. et al., (2015); Li, H. L. et al., (2015);

Ousterout, D. G. et al., (2015a); Ousterout, D. G. et al., (2015b))

Epidermolysis bullosa (Osborn, M. J. et al., (2013); Sebastiano, V. et al., (2014))

Leber Congenital Amaurosis type 10 (Maeder, M. L. et al., (2015))

Cystic fibrosis (Schwank, G. et al., (2013); Crane, A. M. et al., (2015); Firth, A. L. et al., (2015); McNeer, N. A. et al.,

(2015))

Antimicrobials (Bikard, D. et al., (2014); Citorik, R. J. et al., (2014); Gomaa, A. A. et al., (2014))

Maeder and Gersbach, Molecular Therapy 2016

Duchenne Muscular Dystrophy

• Occurs 1/3500 male births

• Debilitating during childhood &

death during 20’s

• Respiratory complications &

cardiac myopathy

• Inherited or spontaneous mutation

to dystrophin

• 79 exons over 2.5 Mb (14 kb

cDNA)

• Cytoskeletal structural protein

• Cell integrity & intracellular

signaling

• No current therapeutic options! Actin

Dystrophin

Glycoprotein

Complex

Extracellular

Matrix

Dystrophin

Restoring Dystrophin Expression around

Exon 44-50 Deletion Hotspot

44 46 47 48 49 50 51 52

44 51 52

45

44 52

Dystrophin mRNA transcript Resulting dystrophin protein

DMD genotype

After correction

Phenotype

Normal

DMD

Mild

• Exon 51 skipping can correct 13% of DMD mutations

• Oligonucleotide-mediated exon skipping is successful in preclinical

models and active in clinical trials (Lancet, N Engl J Med, March 2011)

• Requires lifelong treatment once a week

• Goal: Restoration by genome editing

stop

Ousterout et al. Molecular Therapy (2013), Molecular Therapy (2014),

Nature Communications (2015) Dave Ousterout

- + + - + + - +gRNA

Cas9 + + + + + + + +

% indels: 5.0 4.0 6.8 9.7 5.3

1 2 3 4 5Target

Exon 51

out-of-frame stop codon

1 2 3 4 5

% deleted: 13.6

unmodifieddeletions

GCTTTGATTTCCCTAGGG..//..CCCACCAGTTCTTAGGCAA

GCTTTGATTTCC..................AGTTCTTAGGCAA (x2)

GCTTTGATT.........................CTTAGGCAA

GCTTTGATTTCC........(+41bp).......CTTAGGCAA

Intron 51Intron 50

CR1

PAM

CR5

PAM

CR1/5 treated

genomic DNA

TCTTAACCATTACCATAG..//..CCCACCAGTTCTTAGGCAAC

TCTTAACCATTACCATAG............AGTTCTTAGGCAAC(x2)

TCTTAACCATTACCA......A........AGTTCTTAGGCAAC

TCTTAACCATTACCATAG.............GTTCTTAGGCAAC

CR2

PAMCR5

PAM Intron 51Intron 50

Intron 50 Intron 51

Intron 50 Intron 51CR2/5 treated

genomic DNA10.5

Genomic DNA

Precise deletion of exon 51 from the genome

**

** *

***

**

Ousterout et al. Nature Comm (2015)

Editing the Dystrophin Gene with CRISPR/Cas9

Exon 51

out-of-frame stop codon

1 2 3 4 5

mRNA

Δ48-50

Δ48-51

Exon 47 Exon 52

Western blot

Deletion of exon 51 from the

genome results in restored

dystrophin expression

Dystrophin

GAPDH



HEK293T

hDMD Mbs

Δ45-55

Δ48-50

GAACCAAACCCACT..//..CCTCGATAGGGGATAA

GAACCAAACCCACT............TAGGGGATAA (x5)

Intron 55Intron 44CR6

PAM

CR36

PAM

Dystrophin

GAPDH

Intron 44 Intron 55

Exon 44 Exon 56

Exon 45-55 deletion

(336,380 bp)

Exon 51 deletion

(~800-1050 bp)

45 46 47 48 49 50 51 52 53 54 5544 56

• Exon 51 skipping can correct 13% of DMD mutations (Phase III trials)

• Skipping 45-55 can correct 62% of DMD mutations (Aartsma-Rus et al., Hum Mutat 2009)

• Multi-exon skipping in preclinical development (Aoki et al., PNAS 2012)

Genomic DNA

mRNA

Protein



Gene Editing In Vivo with rAAV

Adeno-associated virus:

• Intramuscular injection of AAV1 approved in Europe (Glybera)

• In preclinical development for delivery of ZFNs to liver for hemophilia by

Shire/Sangamo (Li et al., Nature 2011, Anguela et al., Blood 2013)

• Systemic delivery of AAV to skeletal and cardiac muscle is possible

(Gregorevic et al., Nat Med 2004; Wang et al., Nature Biotechnol 2005;

Asokan et al, Nat Biotechnol 2010)









Deletion of exon 23 in the mdx mouse

Nelson et al. Science (2016)









SaCas9: Feng Zhang, Broad/MIT

Ran, Cong, Yan et al., Nature 2015

AAV: Aravind Asokan, UNC-CH

Chris Nelson










Chris Nelson

gDNA and mRNA PCR

Western blot and IHC

8 weeks

i.m. injection of AAV8 (1E12 vg)

into tibialis anterior muscle


Multiplexed Cas9 Deletes

Exon 23 from the Genome

182016 CRS Annual Meeting –

Edinburg Scotland

22 23 24 22 23 24

1 2

NHEJ


Genomic Deletion Removes

Exon 23 from the Transcript

22 23 24 22 24


Exon 23 Removal Salvages

Protein Expression


Edinburg Scotland Nelson et al. Science (2016)

Dystrophin Localizes into the

Sarcolemma in ~70% of Fibers


Edinburg Scotland

21scale bar - 100 µm


Dystrophin Restoration

Improves Muscle Function

22

specific

twitch force (Pt)

specific

tetanic force (Pt)Repeated eccentric

contraction


Gene Editing in the Heart

Intravenous AAV deliveryNelson et al. Science (2016)

gDNA mRNA

Related Studies

Summary• Genome editing for Duchenne Muscular

Dystrophy

• Multiple strategies for correcting reading frame

• Restoration of dystrophin expression in

myoblasts from DMD patients

• No toxicity and limited off-target activity

• Robust gene editing, dystrophin restoration,

and improved function following in vivo delivery

• Challenges:• Safety

• Immunogenicity

• Delivery & Efficiency

• Progenitor cells

• General tool for science and medicine

What’s next?

Sayyed K. Zaidi et al. Mol. Cell. Biol. 2010;30:4758-4766

Epigenetics

• Applications for controlling enhancer activity

– Modulate multiple genes by targeting a single locus

– Enhancers regulate development and disease

• Differential enhancer profiles in different cell types

• “Super-enhancers” form near oncogenes in cancer

• GWAS studies show association between enhancer SNPs and

disease

Loven et al., Cell, 2013; Hnisz et al., Cell, 2013

Technology for Perturbing Gene

Regulatory Elements

Epigenetics and Gene Regulation

Ong and Corces, 2011

RNA-Guided CRISPR/Cas9 Nucleases

A T C GC G AG A T C G A TC CA T C GC C GGT

gRNA

5′

GA G

Cas9

3′

Cas9

gRNA

Nishimasu et al., Cell 2014

Epigenome Editing with CRISPR/Cas9


gRNA

5′

GA G

dCas9

3′

dCas9 --

gRNA

Effector

domain

Effector domains:

• Nuclease

• Recombinase

• Transcriptional activator or repressor

• DNA methylation

• Histone modificationNishimasu et al., Cell 2014

Jinek et al., Science 2012

Qi et al., Cell 2013

Gilbert et al., Cell 2013



gRNA

5′

GA G

dCas9

3′

Nishimasu et al., Cell 2014

Unresolved questions:

1) Repression of regulatory elementsKearns et al., Nat Methods (2015)

2) Mechanisms of repression

3) Specificity of epigenome editing

4) Spreading of epigenome editing

Qi et al., Cell 2013

Gilbert et al., Cell 2013

KRAB

Repressor

domain

Jinek et al., Science 2012

• DNAse Hypersensitive site in globin gene locus

– HS2 a potent enhancer of the globin locus

– Mutation in LCR reduces globin locus expression

– Contains transcription factor binding sites necessary

for enhancer function

Model system: HS2 enhancer

Pratiksha Thakore

Globin expression following lentiviral

delivery of HS2-targeted dCas9-KRAB

0

0.2

0.4

0.6

0.8

1

1.2

1.4

NogRNA

IL1RN Cr2 Cr4 Cr7 Cr10

Rela

tive

mR

NA

Exp

ressio

n AAA A,B

B,C

C,DD

E

F FF

F

0

0.2

0.4

0.6

0.8

1

1.2

1.4

NogRNA


AA

A,B A,B A,B

B,C

CC,D

D,E

E E E

0

0.2

0.4

0.6

0.8

1

1.2

1.4

NogRNA


A

A,B

AA

AA,B

B,CC,D

C,D

DD

D

hUbc dCas9NLS NLSFLAG KRAB T2A Puro5’ LTR 3’ LTR

hU6gRNA

dCas9 dCas9-KRAB

HBE1 HBG HBB

Thakore et al., Nature Methods (2015)

Cr10

Cr4

HS2 enhancer

FosL1GATA

dCas9-KRAB Interferes with

Transcription Factor Binding

0

0.2

0.4

0.6

0.8

1

1.2

dCas9-KRAB

only

dCas9-KRAB

+ Cr4

dCas9-KRAB

+ Cr10

HS

2/G

AP

DH

GATA2

0

0.2

0.4

0.6

0.8

1

1.2

dCas9-KRAB

only

dCas9-KRAB

+ Cr4

dCas9-KRAB

+ Cr10

HS

2/G

AP

DH

FosL1

HS2 enhancer

FosL1

GATA

Cr10

Cr4

** * *

Guided dCas9-KRAB Interferes with

Transcription Factor Binding

dCas9-KRAB + Cr4

vs dCas9-KRAB only

dCas9-KRAB + Cr10

vs dCas9-KRAB only

Highly Specific Gene Knockdown

RNA-seq

Mean Normalized RNA-seq Signal

log

2(F

old

Ch

an

ge)

Mean Normalized RNA-seq Signal

log

2(F

old

Ch

an

ge)


dCas9-KRAB + Cr4 vs dCas9-KRAB only dCas9-KRAB + Cr10 vs dCas9-KRAB only

dCas9-KRAB only

dCas9 + Cr4

dCas9-KRAB + Cr4

dCas9-KRAB + Cr10

dCas9 + Cr10

K562 DNase HS

HBB HBD HBBP1 HBG2 HBE1Cr10

Cr4HBG1

Highly Specific DNA Binding

ChIP-seq: dCas9-KRAB (HA epitope)

Mean Normalized ChIP-seq Signal

log

2(F

old

Ch

an

ge)


log

2(F

old

Ch

an

ge

)

dCas9-KRAB + Cr10 vs dCas9-KRAB only


log

2(F

old

Ch

an

ge

)

chr11:5301749-5302337 (Cr4 target, HS2)

chr11:5301749-5302337 (Cr10 target, HS2)

Highly Specific Epigenome Editing


ChIP-seq: H3K9me3

chr11:5301862-5302715 (Cr4 target site, HS2)

chr11:5299712-5300301

chr11:5305857-5306185 (HS3)

chr11:5304696-5305098


log

2(F

old

Ch

an

ge)


log

2(F

old

Ch

an

ge)

dCas9-KRAB only

dCas9 + Cr4

dCas9-KRAB + Cr4

dCas9-KRAB + Cr10

dCas9 + Cr10

K562 DNase HSHBB HBD HBBP1 HBG2 HBE1

Cr10

Cr4HBG1

Epigenetic Silencing with dCas9-KRAB

DNase-Seq: Genome wide measure of chromatin accessibility

chr11:5276005-5276305 (HBG2)

chr11:5271045-5271435 (HBG1)

chr11:5270905-5271205 (HBG1)

chr11:5301930-5202230 (Cr4 target, HS2)

chr11:5301930-5202230 (Cr4 target, HS2)

chr11:5275809-5276109 (HBG2)

chr11:5305942-5306243 (HS3)

chr11:5305806-5306106 (HS3)

chr11:5301930-5302230 (HS2)

chr11:5305806-5306106 (HS3)

chr11:5305943-5306243 (HS3)

chr11:5275809-5276109 (HBG2)

chr11:5279828-5280129

chr11:5301764-5302064 (Cr10 target, HS2)



log

2(F

old

Ch

an

ge)


log

2(F

old

Ch

an

ge)


Epigenetic Silencing with dCas9-KRAB

DNase-Seq: Genome wide measure of chromatin accessibility

log2(Fold Change)

p-v

alu

e

p-v

alu

e


log2(Fold Change)

chr11:5301930-5302230 (HS2)

chr11:5301764-5302064 (Cr10 target, HS2)

chr11:5305806-5306106 (HS3)

chr11:5305943-5306243 (HS3)

chr11:5279828-5280129

chr11:5275809-5276109 (HBG2)


chr11:5305806-5276106 (HS3)

chr11:5305942-5306243 (HS3)

chr11:5301930-5202230 (Cr4 target, HS2)

chr11:5275809-5276109 (HBG2)

chr11:5276005-5276305 (HBG2)

chr11:5301930-5202230 (Cr4 target, HS2)

chr11:5271045-5271435 (HBG1)

chr11:5270905-5271205 (HBG1)


Histone acetylation regulates the genome

Genomic Accessibility

Histone AcetylTransferase

(HDACs/HMTs)(HATs)

Tessarz and Kouzarides, Nat Rev Mol Cell Biol, 2014

Verdin and Ott, Nat Rev Mol Cell Biol, 2015

Genomic Activation Signals

Unique chromatin signatures are associated with

transcriptional and epigenetic states

Roadmap Epigenomics Consortium, Nature, 2015

Active TSS

Promoters

Enhancers

Histone Acetylation


gRNA

5′

GA G

dCas9

3′

Acetyl-

transferase


Targeted CRISPR/Cas9-Based

Acetyltransferase

• Targeted epigenome editing and gene activationHilton et al., Nature Biotechnol (2015)

Isaac Hilton


Acetyltransferase

• Activation of target genes from enhancers by acetylation

Hilton et al., Nature Biotechnol (2015)


Acetyltransferase


• Activation of target genes from enhancers by acetylation


Acetyltransferase



Acetyltransferase

• Acetylation at target promoter

following enhancer activationHilton et al., Nature Biotechnol (2015)


Acetyltransferase

• Highly specific gene activation by targeted epigenome

editing


The Expanding Epigenome Editing Toolbox

Thakore et al., Nature Methods 2016

• Applications for controlling enhancer activity

– Modulate multiple genes by targeting a single locus

– Enhancers regulate development and disease

• Differential enhancer profiles in different cell types

• “Super-enhancers” form near oncogenes in cancer

• GWAS studies show association between enhancer SNPs and

disease

Loven et al., Cell, 2013; Hnisz et al., Cell, 2013

Technology for Perturbing Gene

Regulatory Elements

Applications of Epigenome Editing

Thakore et al., Nature Methods 2016

Summary

• CRISPR/Cas9 for gene regulation and targeted

epigenome editing

• Genome-wide specificity of regulation, binding, and

remodeling

• Interrogating function of epigenetic marks

• Acetylation as causal for gene activation

• Genetic reprogramming via targeted gene activation

• Applications in functional epigenomics

• Mapping GWAS hits and annotating enhancer function

• Other epigenetic modifiers – DNA methylation and

histone modifications

Gersbach LabShaunak Adkar

Joe Bellucci, PhD

Josh Black

Malathi Chellapan

Rui Dai

Matt Gemberling, PhD

Isaac Hilton, PhD

Liad Holtzman

Hunter Hutchinson

Tyler Klann

Dewran Kocak

Jennifer Kwon

Feimei Liu

Sarina Madhavan

Christopher Nelson, PhD

Matt Oliver

Adrian Pickar, PhD

Adrianne Pittman

Jay Rathinavelu

Jacqueline Robinson-Hamm

Pratiksha Thakore, PhD

CollaboratorsFarshid Guilak (Duke/WashU)

Dongsheng Duan (U Missouri)

Feng Zhang (Broad/MIT)

Aravind Asokan (UNC-CH)

Jacques Tremblay (U Laval)

Tim Reddy (Duke)

Greg Crawford (Duke)

Kam Leong (Duke/Columbia)

AlumniJonathan Brunger, PhD

Tyler Gibson, PhD

Katie Glass, PhD

Ami Kabadi, PhD

Adim Moreb

David Ousterout, PhD

Pablo Pérez-Piñera, MD, PhD

Lauren Polstein, PhD

Funding: NIH Director’s New Innovator Award (DP2OD008586), NIH (R01DA036865, R01AR069085, T32GM008555, U01HG007900, UH3TR000505, P30AR066527, R21AR065956, R21AR067467, R21DA041878, R03AR061042), NSF CAREER Award (CBET-1151035), Muscular Dystrophy Association, CDMRP (MD140071), The Hartwell Foundation, March of Dimes Foundation, American Heart Association, Nancy Taylor Foundation, Duke-Coulter Partnership, Duke Clinical and Translational Science Award

Thank You

Nuclease-inactive dCas9 is a versatile platform for

editing transcription and chromatin

Targeted Nuclease (Cas9) Targeted Genome Binding (dCas9)

Platform :

Hilton et al, Nat. Biotechnol, 2015

Methods to precisely modulate endogenous

transcription and chromatin are needed

Targeted Effector

Molecules

Endogenous Regulatory Element Validation

Objective: Engineer methods to characterize targeted acetylation

of chromatin at endogenous human regulatory elements

H3K27ac found at

Active and “Poised”

Regulatory Elements

Causal?

crispr/cas9 technology for gene therapy and epigenome …...crispr/cas9 technology for gene therapy...

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