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 Biologycmoraes@med.miami.edu

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