numerous therapeutic avenues for fshd...dna hypomethylation of shortened 4q35 fshd2: dominant...
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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
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