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
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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
Ousterout et al. Nature Comm (2015)
Editing the Dystrophin Gene with CRISPR/Cas9
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
Ousterout et al. Nature Comm (2015)
Editing the Dystrophin Gene with CRISPR/Cas9
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)
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)
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)
SaCas9: Feng Zhang, Broad/MIT
Ran, Cong, Yan et al., Nature 2015
AAV: Aravind Asokan, UNC-CH
Chris Nelson
Nelson et al. Science (2016)
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)
Chris Nelson
gDNA and mRNA PCR
Western blot and IHC
8 weeks
i.m. injection of AAV8 (1E12 vg)
into tibialis anterior muscle
Nelson et al. Science (2016)
Multiplexed Cas9 Deletes
Exon 23 from the Genome
182016 CRS Annual Meeting –
Edinburg Scotland
22 23 24 22 23 24
1 2
NHEJ
Nelson et al. Science (2016)
Genomic Deletion Removes
Exon 23 from the Transcript
22 23 24 22 24
Nelson et al. Science (2016)
Exon 23 Removal Salvages
Protein Expression
202016 CRS Annual Meeting –
Edinburg Scotland Nelson et al. Science (2016)
Dystrophin Localizes into the
Sarcolemma in ~70% of Fibers
2016 CRS Annual Meeting –
Edinburg Scotland
21scale bar - 100 µm
Nelson et al. Science (2016)
Dystrophin Restoration
Improves Muscle Function
22
specific
twitch force (Pt)
specific
tetanic force (Pt)Repeated eccentric
contraction
Nelson et al. Science (2016)
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
A T C GC G AG A T C G A TC CA T C GC C GGT
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
Epigenome Editing with CRISPR/Cas9
A T C GC G AG A T C G A TC CA T C GC C GGT
gRNA
5′
GA G
dCas9
3′
Nishimasu et al., Cell 2014
Unresolved questions:
1) Repression of regulatory elementsKearns et al., Nat Methods (2015)
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)
Mean Normalized ChIP-seq Signal
log
2(F
old
Ch
an
ge
)
dCas9-KRAB + Cr10 vs dCas9-KRAB only
Mean Normalized ChIP-seq Signal
log
2(F
old
Ch
an
ge
)
chr11:5301749-5302337 (Cr4 target, HS2)
chr11:5301749-5302337 (Cr10 target, HS2)
Highly Specific Epigenome Editing
dCas9-KRAB + Cr4 vs dCas9-KRAB only dCas9-KRAB + Cr10 vs dCas9-KRAB only
ChIP-seq: H3K9me3
chr11:5301862-5302715 (Cr4 target site, HS2)
chr11:5299712-5300301
chr11:5305857-5306185 (HS3)
chr11:5304696-5305098
Mean Normalized ChIP-seq Signal
log
2(F
old
Ch
an
ge)
Mean Normalized ChIP-seq Signal
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)
dCas9-KRAB + Cr4 vs dCas9-KRAB only dCas9-KRAB + Cr10 vs dCas9-KRAB only
Mean Normalized ChIP-seq Signal
log
2(F
old
Ch
an
ge)
Mean Normalized ChIP-seq Signal
log
2(F
old
Ch
an
ge)
Thakore et al., Nature Methods (2015)
Epigenetic Silencing with dCas9-KRAB
DNase-Seq: Genome wide measure of chromatin accessibility
log2(Fold Change)
p-v
alu
e
p-v
alu
e
dCas9-KRAB + Cr10 vs dCas9-KRAB only
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)
dCas9-KRAB + Cr4 vs dCas9-KRAB only
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)
Thakore et al., Nature Methods (2015)
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
A T C GC G AG A T C G A TC CA T C GC C GGT
gRNA
5′
GA G
dCas9
3′
Acetyl-
transferase
Epigenome Editing with CRISPR/Cas9
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Targeted epigenome editing and gene activationHilton et al., Nature Biotechnol (2015)
Isaac Hilton
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Targeted epigenome editing and gene activationHilton et al., Nature Biotechnol (2015)
Isaac Hilton
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Targeted epigenome editing and gene activationHilton et al., Nature Biotechnol (2015)
Isaac Hilton
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Targeted epigenome editing and gene activationHilton et al., Nature Biotechnol (2015)
Isaac Hilton
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Targeted epigenome editing and gene activationHilton et al., Nature Biotechnol (2015)
Isaac Hilton
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Activation of target genes from enhancers by acetylation
Hilton et al., Nature Biotechnol (2015)
Targeted CRISPR/Cas9-Based
Acetyltransferase
Hilton et al., Nature Biotechnol (2015)
• Activation of target genes from enhancers by acetylation
Targeted CRISPR/Cas9-Based
Acetyltransferase
Hilton et al., Nature Biotechnol (2015)
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Acetylation at target promoter
following enhancer activationHilton et al., Nature Biotechnol (2015)
Targeted CRISPR/Cas9-Based
Acetyltransferase
• Highly specific gene activation by targeted epigenome
editing
Hilton et al., Nature Biotechnol (2015)
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