Targeting Disease-Causing Defects of the Mitochondrial Genome with Engineered Mitochondrial...
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![Page 1: Targeting Disease-Causing Defects of the Mitochondrial Genome with Engineered Mitochondrial Nucleases Targeting Disease-Causing Defects of the Mitochondrial.](https://reader031.fdocuments.us/reader031/viewer/2022032105/56649d355503460f94a0c63c/html5/thumbnails/1.jpg)
Targeting Disease-Causing Defects of the Mitochondrial Genome with
Engineered Mitochondrial Nucleases
Carlos T. Moraes
Esther Lichtenstein Professor in NeurologyUniversity of Miami Miller School of Medicine
Dept. of Neurology and Cell [email protected]
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mtDNA Heteroplasmy is Important in Defining Cellular Health
heteroplasmy homoplasmyhomoplasmy
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Most patients have a mixture of mutated and wild-type mtDNA(mtDNA heteroplasmy)
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HOW CAN WE ALTER mtDNA HETEROPLASMY?
Wild-type Mutated
*
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Mito-RERE
mito-targeting sequence
polyA
RE
Targeting Restriction Endonucleases to the mitochondrial matrix to promote mtDNA double strand breaks (DSB) and
degradation of mtDNA
RE mtDNA
mtDNA
mtDNA
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A mouse model for mtDNA haplotype differential recognition by a mito-RE
BALB/NZB mtDNA heteroplasmic mouse cells
ApaLI Sites in mtDNA
BALB 1
NZB 0
Expression of Mito-ApaLI
ApaLIC8 HA
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Examine tissues after:6 weeks3 months6 months
AAV9-mito-ApaLI-HA
2-3 days old neonates
Uses of Heteroplasmic MiceTargeting Muscle – AAV9
IP or IV InjectionsNZB
Balbc
ApaLIC8 HA
ApaLI
AAV9-Alkalyne Phosphatase
Gastro
cnem
ius
Quadr
iceps
Soleus
Tib.
Ant
.
Gastro
cnem
ius
Quadr
iceps
Soleus
Tib.
Ant
.
ApaLI-HA
MW
AP Mito-ApaLI
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G Q S BI G Q S BI U
AAV9-AP
NZB mtDNA
Balb mtDNA
AAV9-mito-ApaLI-HA
mtDNA heteroplasmy changes 6 weeks after IP injections of AAV-mito-ApaLI-HA
BI=Before Injection (ear biopsy)G=GastrocnemiusQ=QuadricepsS=Soleus
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New Tools for Genome Editing[ZFN, TALEN, CRISPR]
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TAL Effector Domains
Developing DNA-binding motifs
with new specificities
T0
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DNA Binding by TAL Effector Proteins
Structure of a TAL effector protein wrapped around a DNA double helix. Image based on data from Mak et al., Science 2012
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TAL Effector Nucleases (TALEN)
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mitoTALEN to Cleave mtDNA Point Mutations
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mitoTALEN to Cleave mtDNA point mutations
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mtDNA Changes 14 Days after Transfection
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Complex I Activity in mitoTALEN treated Cells
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Mutant mtDNA elimination
Is not “EDITING”
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IMS
matrix
succinate fumarate
Cyt c
The Oxidative Phosphorylation System
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mtDNA: CaucasiannDNA: Japanese
Is incompatibility between nuclear
DNA and mtDNA a health problem?
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Kinetics of mtDNA Heteroplasmy Shift
NZB Balbc
ApaLI
ApaLIC8 HA