Genome Editing for Duchenne Muscular Dystrophy · Duchenne Muscular Dystrophy Nelson and Gersbach,...

Genome Editing for Duchenne Muscular Dystrophy

Charles A. Gersbach, PhDDepartment of Biomedical Engineering

Department of SurgeryCenter for Genomic and Computational Biology

Duke University

June 28, 2019Parent Project Muscular Dystrophy

Orlando, FL

Sarepta Therapeutics – sponsored research, consultancy

These relationships are managed by Duke University.

Disclosures

Gene Editing

Duchenne Muscular Dystrophy

Nelson and Gersbach, Muscle Gene Therapy (2018)

Duchenne Muscular Dystrophy

Nelson and Gersbach, Muscle Gene Therapy (2018)

• Why somatic cell gene editing for Duchenne?

• Clinical need

• Potential to create larger, perhaps full-length dystrophin protein

• Potential to edit genes in satellite cells

• Potential for long-term dystrophin restoration• Multinucleated target cells means low level editing may be sufficient

• Correctable by exon excision

• Hotspot mutation regions can be addressed for large numbers of patients

Genome Editing with Engineered Nucleases

Nelson and Gersbach, Annual Review Chem Biol Eng (2016)

Targeted insertionsSplice site disruption

ORre-framing

Exon excision

Targeted breaks in DNA stimulate natural DNA repair pathways

(Image: McGovern Institute for Brain Research at MIT)

CRISPR/Cas9



Targeted insertions Exon excision


Splice site disruptionOR

re-framing

Single-Cut Reframing for DMDWT dystrophin protein

WT dystrophin46 47 49 50 51 52 53 54484544 5655

Deletion of exons 48-50DMD – out of frame, no protein

BMD – truncated, partially functional protein

Deletion of exons 48-51; applicable to ~13% patients

Ousterout et al. Molecular Therapy (2013), Molecular Therapy (2014), Nature Communications (2015)

46 47 51 52 53 544544 5655

46 47 52 53 544544 565551*

Dave Ousterout

Single-Cut Reframing for DMDWT dystrophin protein

WT dystrophin46 47 49 50 51 52 53 54484544 5655





46 47 51 52 53 544544 5655

46 47 52 53 544544 565551*

Dystrophin

GAPDH

WT DMDDMD +TALEN

WT CTCAGACTGTTACTCTGGTGACACAA shift (frame)32 CTCAGACTGTT----TGGTGACACAA -4 (+2)40 CTCAGACTGTTACTCTG-TGACACAA -1 (+2)67 CTCAGACTG---------TGACACAA -9 (+3)96 CTCAGACTGTTACTC-GGTGACACAA -1 (+2)106 CTCAGACTGTTACT-----GACACAA -5 (+1)127 CTCAGACTGTTACTCTG---ACACAA -3 (+3)141 CTCAGACTGTTACT---GTGACACAA -3 (+3)145 CTCAGACTGTTACTCTG---ACACAA -3 (+3)

Single-Cut Reframing for DMD





ORre-framing

Exon excision



WT dystrophin proteinWT dystrophin

46 47 49 50 51 52 53 54484544 5655





46 47 51 52 53 544544 5655

46 47 52 53 544544 5655


WT dystrophin proteinWT dystrophin

46 47 49 50 51 52 53 54484544 5655




Why exon deletion?• NHEJ > HDR• Predictable and reproducible protein product• Large intronic regions allow for gRNA optimization• Multi-exon skipping can increase applicability

Ousterout et al. Molecular Therapy (2013), Molecular Therapy (2014), Nature Communications (2015) Dave Ousterout

46 47 51 52 53 544544 5655

46 47 52 53 544544 5655


DMD patient cells mdx mouse hDMD mouse

Ousterout et al., Nature Communications (2015), Molecular Therapy (2013, 2015) Nelson et al., Science (2016)

Robinson-Hamm et al., unpublished


Realizing the promise of gene editing:

• Immunogenicity

• Durability

• Satellite Cell Targeting

• Restoration of a Full-Length Dystrophin

Host Immune Response to CRISPR/Cas9

0.1

1

10

100

1000

AdultIM IV 8wk 1yr 8wk 1yr

Neonate UTIV 8wk

Serum

SaC

as9 I

gG (µ

g/mL)

A)

IV AAV8IM AAV8

IV AAV9Untreated

** * **

SFC

per2

.5 x

10 c

ells

5

0

10

20

30B)

Adult

NeonateUT

*

AAV9: - + +

⍺-Cas9 antibody response

Nelson et al., Nature Medicine (2019)

Host Immune Response to CRISPR/Cas9

• ⍺-Cas9 response following treatment of adults but not neonates• Used ubiquitous promoter – tissue-restricted promoters may help

0.1

1

10

100

1000


Neonate UTIV 8wk

Serum

SaC

as9 I

gG (µ

g/mL)

A)

IV AAV8IM AAV8

IV AAV9Untreated

** * **

SFC

per2

.5 x

10 c

ells

5

0

10

20

30B)

Adult

NeonateUT

*

AAV9: - + +0.1

1

10

100

1000


Neonate UTIV 8wk

Serum

SaC

as9 I

gG (µ

g/mL)

A)

IV AAV8IM AAV8

IV AAV9Untreated

** * **

SFC

per2

.5 x

10 c

ells

5

0

10

20

30B)

Adult

NeonateUT

*

AAV9: - + +

⍺-Cas9 antibody response ⍺-Cas9 T cells




• Immunogenicity

• Durability



Persistence of Editing in Skeletal and Cardiac Muscle



Significant increase in DNA editing frequency from 8 weeks to 1 year




Significant increase in editing frequency from 8 weeks to 1 year



• Immunogenicity

• Durability



Satellite Cells are the Stem Cells of Skeletal Muscle

Does AAV transduce satellite cells in vivo?

Does CRISPR edit satellite cells in vivo?

Does satellite cell editing facilitate long-term

dystrophin restoration?

AAV-CRISPR Gene Editing of Satellite Cells

Kwon et al., unpublished

AAV8-CRISPR

GFP+4.93%

Pax7 Exon 1 nGFP

Jennifer Kwon


AAV8-CRISPR

GFP+4.93%

Pax7 Exon 1 nGFP


Jennifer Kwon


AAV8-CRISPR

GFP+4.93%

Pax7 Exon 1 nGFP

mouse 1

GFP:

mouse 2 mouse 3

+ - + - + -

∆23


Vance et al., 2015

Diverse AAV Serotypes with Various Tissue Tropisms


Systematic Assessment of AAV Transduction of Satellite Cells

Pax7-nGFP +/-Ai9 +/-

mdx +/0

CAG

Rosa26

Pax7 Exon 1 nGFP

STOP tdTomatoCAGtdTomato+

GFP+

AAV: CMV-CRE

8wks

tdTomato


Efficient Genetic Labelling of Satellite Cells by Multiple AAV Serotypes

Local AAV Delivery(4.72E+11 vg/TA muscle)

AAV9

AAV6.2AAV8

AAV1AAV2

AAV50

20

40

60

80

%Td

Tom

ato

of G

FP+

Recombination Efficiency of Pax7-GFP+ cells

n=5, mean + SEM; 8 weeks post-injections


Local AAV Delivery(4.72E+11 vg/TA muscle)

AAV9

AAV6.2AAV8

AAV1AAV2

AAV50

20

40

60

80

%Td

Tom

ato

of G

FP+

Recombination Efficiency of Pax7-GFP+ cells

n=5, mean + SEM; 8 weeks post-injections

Quad TA

Diaphra

gm

Soleus

Tricep

0

20

40

60

%T

dT

om

ato

of G

FP

+

AAV9 Systemic Recombination Efficiency of Pax7-GFP+ cells

A

B

C

D

E A

B

C

DE

A

BC

D

E

AB

C

D

E

A

B

C

D

E

Systemic AAV9 Delivery(2E+12 vg/mouse)



Regenerative Potential of CRISPR-Edited Satellite Cells

Dystrophin/Laminin

Intron 22 Intron 23

Δ23

mouse: 1L 2L 3L 1R 2R 3R Pos ctrl

Deletion PCR at Dmd locus

Serial Engraftment Strategy:Pax7-nGFPmdx donor

AAV9-CRISPRs

Isolate GFP+ cells

pre-injuredmdx host

Uninjected Injected



• Immunogenicity

• Durability






ORre-framing

Exon excision

Full Length Dystrophin Restoration by Targeted Integration

Adrian Pickar Oliver, PhD


Pickar-Oliver et al., unpublished

Expected size:300bp



Expected size:300bp

hDM

D

hDMD∆52PB

S g7-Ex52 (1:1) g7-Ex52 (1:5)g12-Ex52 (1:1) g12-Ex52 (1:5)PB

S

500bp300bp



cDNA:

Expected size:300bp

hDM

D

hDMD∆52PB

S g7-Ex52 (1:1) g7-Ex52 (1:5)g12-Ex52 (1:1) g12-Ex52 (1:5)PB

S

500bp300bp

hDM

D

hDMD∆52

PBS g7-Ex52 (1:1) g7-Ex52 (1:5)

PBS

460 kD-

41 kD-

← Dystrophin

← GAPDH



cDNA:

Protein:

Summary• Genome editing for DMD typically focuses on removing gene

segments to restore functional, truncated dystrophin

• Single gene editing strategy can treat ~50% of DMD patients

• Robust anti-Cas9 host immune response that resolves without intervention

• In vivo CRISPR-based genome editing restores long-term dystrophin expression in many studies with no reported adverse effects

• Unintended genomic outcomes, including AAV integration, into on-target and off-target sites

• Full-length dystrophin restoration is possible but requires patient-specific approaches

• Additional research required to understand implications of immune response, long-term presence of delivery vectors, and alternative genome modifications

Gersbach LabJosh BlackJosie Bodle, PhDLexi BoundsJoel BohningKaren Bulaklak, PhDGabe Butterfield, PhDBrian Cosgrove, PhDHeather DanielsClaire EngstromAdarsh EttyreddyMatt Gemberling, PhDKylie GilcrestVeronica GoughLiad HoltzmanNahid Iglesias, PhDAriel KantorTyler KlannDewran KocakJennifer KwonBill Majoros, PhDSean McCutcheonChristopher Nelson, PhDAdrian Pickar, PhDAdrianne PittmanMadeleine SittonAlan SuAshish Vankara

CollaboratorsAravind Asokan (Duke)Joel Collier (Duke)Tim Reddy (Duke)Greg Crawford (Duke)Nenad Bursac (Duke)Rodolphe Barrangou (NC State)Raluca Gordan (Duke)Anne West (Duke)Dongsheng Duan (U Missouri)Chase Beisel (NC State)Yong-hui Jiang (Duke)Xiling Sheng (Duke)

AlumniShaunak AdkarJoe Bellucci, PhDJonathan Brunger, PhDTyler Gibson, PhDIsaac Hilton, PhD (Rice U)Ami Kabadi, PhDAdim MorebDavid Ousterout, PhDPablo Pérez-Piñera, PhD (UIUC)Lauren Polstein, PhDJacqueline Robinson-Hamm, PhDPratiksha Thakore, PhD

Funding: Paul G. Allen Frontiers Group, Open Philanthropy, NIH, NSF, Muscular Dystrophy Association, Gilbert Foundation, Department of Defense, Thorek Memorial Foundation, The Hartwell Foundation, March of Dimes Foundation, American Heart Association, Nancy Taylor Foundation, Duke-Coulter Partnership, Duke Clinical and Translational Science Award, Sarepta Therapeutics, Locus Biosciences, Element Genomics, Levo Therapeutics

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