Numerous therapeutic avenues for FSHD

Himeda et al. (2015) Antiox Redox Signaling

“Treasure your exceptions.”Thomas Hunt Morgan

Epigenetics

Ø Context-dependent sequence independent gene expressionØ Stable yet dynamic à Cellular memoryØ Can be influenced by the environment (diet, aging, etc…)

FSHD genetics are complex

Himeda et al. (2014) Antiox Redox Signaling

FSHD is primarily an epigenetic disease

High variability within the clinical and genetic populationà Presentation, progression, severity, asymmetry,

extramuscular, and age of onset

4qter4qA

FSHD24qter

4qA

FSHD14

4

De Greef et al. (2009) Human Mutation

Healthy4qA

4qter4

More euchromatic More heterochromatic

All types of FSHD are linked to epigenetic status of 4q35 D4Z4

FSHD1: Dominant deletions at 4q35 D4Z4 arrayApparently low penetranceDNA Hypomethylation of shortened 4q35

FSHD2: Dominant inactivating mutations in SMCHD1--ATPase chromatin remodeling protein-- account for ~85% of FSHD2 à more mutations

DNA Hypomethylation of 4q35 and 10q26 arrays

IFSHD: Infantile form of FSHD1 or FSHD2, much more severeDNA Hypomethylation of FSHD1 or 2

Epigenetic Gene andGenome Regulation

“The structural adaptation of chromosomal regions so as to register, signal or perpetuatealtered activity states” - Adrian Bird

Keys: DNA sequence independentContext dependentStable/HeritableCellular MemoryDynamic/reversibleResponsive

Chromatin: DNA, histones, non-histone proteins, RNA

Epigenetics: Nurture (Environment) vs. Nature

Heritable changes in gene activity that do not involve alterations to the genetic code

Nature: Genetics Nurture: Epigeneticsvs.

Passed on by Mom and Dad

Affected by:• Mutagens• Mistakes

Passed on by Mom and Dad

Affected by:• Stress• Sleep• Diet

Genetically identical Genetically identical,epigenetically different

• Prenatal care• Environment

Epigenetic differences in genetically ~ equivalent people (i.e. identical twins) can have profound effects

Waterland & JirtleWaterland and Jirtle (2003) MCB 23:5293

“Epialleles”

Epigenetic differences can have profound long-term health consequences

AIAP allele,methylated

AIAP allele,unmethylated

Genetically identicalEpigenetically different Affects long-term health

à heritable?

Brown, normal

Yellow, obese, spontaneous tumors

Epialleles

Waterland and Jirtle, Mol Cell Biol, 2003

4qterFSHD1

4qA

FSHD24qter

4qA

=HypermethylatedCpGs=moreheterochromatic

=HypomethylatedCpGs=moreeuchromatic

Healthy4qA

4qter

4qter4qA

FSHD epigenetics dictate disease

FSHD1asymptomatic

4

4

4

4

N= 1-10RU

N= 11-26 RU

N= 5-10RU

Epigenetic Therapies

FSHD Euchromatic D4Z4 Chromatin structure

Rett Syndrome Reactivation of silenced X chromosome

Fragile X Syndrome Reactivation of FMR1 genePrader-Willi SyndromeLeukemia Reactivation of tumor Cancer suppressors

Duchenne MD Induction of compensatory Myotonic Dystrophy genes

Emery-Driefuss MD Nuclear architecture

Disease Epigenetic target ApproachesSmall molecule inhibitorsCRISPR/Cas9/dCas9

Heterochromatic structure

Small molecule inhibitors

Small molecule inhibitors

Small molecule inhibitors

Small molecules

???

PAS=polyadenylationsiteNP=non-permissive=translationstopP=permissive*

*PAS

Exon1 E2 E3(P)

AAAAAAA -StablepolyAmRNA-DUX4proteinproduced-Cytotoxic

*Exon1 E2 E3(NP)

-UnstablemRNA-DUX4proteinnotmade-Nontoxic

Non-permissive

Permissive

D4Z4

D4Z4

DUX4-fl

“B” and NP “A”-typesubtelomeres

Typical “A”-typesubtelomeres

PAS = polyadenylation site = translation stop*

*No PAS

Exon1 E2 E3

degradeddegraded

NonpermissiveHealthy

*PAS

Exon1 E2 E3

AAAAAAA DUX4-FL+++++

4qA permissiveFSHD

DUX4-fl expression is dependent upon the PAS and FSHD/healthy status

*

*PAS

Exon1 E2 E3

AAAAAAADUX4-FL

+/-----4qA permissiveHealthy *

No DUX4

Therapeutic approaches to FSHD

Himeda et al. (2015) Antiox Redox Signaling

Epigenetic regulation is a therapeutic target for FSHD

Small molecule inhibitors

CRISPR/Cas9 CRISPRi/dCas9-KRABMorpholinos/PMOs/shRNAs/miRNAs

Anti-inflammatory Myostatin inhibition

Therapeutic approaches to FSHD

CRISPR-Cas9 and dCas9 in muscular dystrophy research and therapeutic development

3’5’

3’3’5’5’

dCas9

CCN

GGN

PAM

DUX4 gene

3’

5’

3’3’5’

5’

sgRNA

Cas9

CCN

GGN

PAM

CRISPR/Cas9 Editing

CRISPR/dCas9 Transcription Modulation

3’5’3’

5’NHEJ

****

Promoters,Enhancers,Gene bodies

KRABRepressor

sgRNA

... ...

... ...

... ...

sgRNA I-44 sgRNA I-55

Cas9 + sgRNA E-51

DUX4

DMD(Δ45-55)

2 3 (PAS)12121D4Z4D4Z4D4Z4

DMD

FSHDAAAAAA

Ch. 4q35

sgRNA E-1 sgRNA E-3sgRNA PAS

***

DMD: dCas9-VP16 upregulated Utrophin mRNA [12]

Cas9 + sgRNAs

A

BDMD mutation hotspot

44 45 46 47 48 5049 51 52 53 54 55 561 79

441 56 79

44 45 46 47 48 5049 52 53 54 55 561 7951

51

Indels

FSHD: dCas9-KRAB repressed DUX4 mRNA [7]

Himeda et al. (2016) Trends Pharmacol.

3’5’

3’3’5’5’

dCas9

CCN

GGN

PAM

DUX4 gene

3’

5’

3’3’5’

5’

sgRNA

Cas9

CCN

GGN

PAM

CRISPR/Cas9 Editing

CRISPR/dCas9 Transcription Modulation

3’5’3’

5’NHEJ

****

Promoters,Enhancers,Gene bodies

KRABRepressor

sgRNA

... ...

... ...

... ...

sgRNA I-44 sgRNA I-55

Cas9 + sgRNA E-51

DUX4

DMD(Δ45-55)

2 3 (PAS)12121D4Z4D4Z4D4Z4

DMD

FSHDAAAAAA

Ch. 4q35

sgRNA E-1 sgRNA E-3sgRNA PAS

***

DMD: dCas9-VP16 upregulated Utrophin mRNA [12]

Cas9 + sgRNAs

A

BDMD mutation hotspot

44 45 46 47 48 5049 51 52 53 54 55 561 79

441 56 79

44 45 46 47 48 5049 52 53 54 55 561 7951

51

Indels

FSHD: dCas9-KRAB repressed DUX4 mRNA [7]

Gilbert et al. (2013) Cell

Himeda et al. (2015) Mol. Therapy

Efficient genome targeting

Therapeutic delivery of CRISPR/Casis challenging

Maeder and Gersbach (2016) Mol Ther 24:430