targeting disease-causing defects of the mitochondrial genome with engineered mitochondrial...
TRANSCRIPT
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]
mtDNA Heteroplasmy is Important in Defining Cellular Health
heteroplasmy homoplasmyhomoplasmy
Most patients have a mixture of mutated and wild-type mtDNA(mtDNA heteroplasmy)
HOW CAN WE ALTER mtDNA HETEROPLASMY?
Wild-type Mutated
*
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
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
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
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
New Tools for Genome Editing[ZFN, TALEN, CRISPR]
TAL Effector Domains
Developing DNA-binding motifs
with new specificities
T0
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
TAL Effector Nucleases (TALEN)
mitoTALEN to Cleave mtDNA Point Mutations
mitoTALEN to Cleave mtDNA point mutations
mtDNA Changes 14 Days after Transfection
Complex I Activity in mitoTALEN treated Cells
Mutant mtDNA elimination
Is not “EDITING”
IMS
matrix
succinate fumarate
Cyt c
The Oxidative Phosphorylation System
mtDNA: CaucasiannDNA: Japanese
Is incompatibility between nuclear
DNA and mtDNA a health problem?
Kinetics of mtDNA Heteroplasmy Shift
NZB Balbc
ApaLI
ApaLIC8 HA