This document summarizes current research on treating mitochondrial DNA disorders through genome editing techniques. It begins with background on mitochondrial genetics and disorders, noting that mutations can occur in either mitochondrial or nuclear DNA. Current therapeutic strategies include preventing transmission of mutations, shifting the ratio of normal to mutated mitochondrial DNA, and directly editing the mitochondrial genome. Genome editing uses enzymes like CRISPR-Cas9 or restriction endonucleases to selectively destroy mutated mitochondrial DNA, potentially reducing symptoms by lowering the mutation load below a threshold level. This targeted destruction of pathogenic mutations while sparing normal DNA may help treat currently incurable mitochondrial disorders.

1. Genome editing to treat
mitochondrial DNA disorders
Maria de los Angeles Avaria MD
Pediatric Neurology
Universidad de Chile

2. BACKGROUND

3. Mitochondrial Proteome
• 13 encoded by mtDNA.
1500 polypeptides
- Encoded in the nucleus
- Synthesized on cytoplasmic ribosomes.
- Imported into mitochondria
> 99%
22
tRNA
2
rRNA

4. Essen,al
for
cellular
func,on.
This
1%
of
total
cellular
DNA,
mtDNA
is
crucial
for
OXPHOS
Electron Transport Chain I II III IV V
Nuclear DNA coding 35 4 8 10 10
mitoDNA coding 7 0 1 3 2
Brockington et al. BMC Genomics 2010 11:203
http://www.biomedcentral.com/
16,500
BP

5. It is estimated that
Risk of developing mt Disease 1 /5000
Prevalence of mtDNA diseases at least 1 /10,000
individuals
1 in 200 women could be a mitochondrial disease
carrier.
involved in complex diseases as Cancer, diabetes and in ageing.
Dysfunctional mitochondria implicated in several neurodegenerative
diseases including Parkinson’s disease
200 mtDNA mutations associated with defined clinical phenotypes
(http://www.mitomap.org)
Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF. The epidemiology of mitochondrial
disorders–past, present and future. Biochim Biophys Acta 2004.
Cree, L.M., Samuels, D.C., Chinnery, P.F.,. The inheritance of pathogenic mitochondrial DNA
mutations. Biochim. Biophys. 2009

6. Combined
data
for
children
and
adults
• ≈
1
in
2000
to
5000
children
will
be
diagnosed
with
mitochondrial
disease
in
their
life,mes.
• (objec,ve
respiratory
chain
deficiency
or
pathogenic
mtDNA
muta,on)
50%
onset
in
the
first
5
years
Naviaux
R.K.
Developing
a
systema,c
approach
to
the
diagnosis
and
classifica,on
of
mitochondrial
disease
Mitochondrion,
4
(2004),
pp.
351–361

7. Endosymbiosis
• Popularized in 1967 by Lynn Sagan Margulis.
• Early eukaryotic cells were invaded by bacteria adapted to oxygen-
rich atmosphere becoming the permanent endosymbionts we call
mitochondria.
• All eukaryotic cells have mitochondrial DNA (mtDNA).
• In the course of evolution most of mtDNA genes have been
transferred to the nuclear genome
Sagan Margulis L. On the origin of mitosing cells. J Theroet Biol. 1967; 14:255–274.

8. MECHANISMS OF DISEASE
mtDNA
muta,ons
Nuclear
muta,ons
affec,ng
mitochondrial
proteins
• Muta,ons
in
nuclear
genes
are
increasingly
becoming
recognized
as
the
major
cause
of
pediatric
mitochondrial
disease
Signaling
• Mt
deple,on
• Mt
mul,ple
dele,ons

9. EPIGENETIC FACTORS
LHON EXAMPLE
• mtDNA homoplasmic mutation ≈ 1 in 300 population
• Blindness ≈ 1 in 20 000
• Males are four to five times more likely to be affected.
• DEAFNESS
m.1555A4G mutation
• Isolated (non-syndromic) deafness
- Spontaneously
- In response to environmental exposure to
aminoglycoside antibiotics.
– Chinnery, P.F.et als Epigenetics, epidemiology and
mitochondrial DNA diseases. Int. J. Epidemiol. 2012

10. France,
debut
de
XIXe
siècle:
Ariane
et
Thésée.
Musée
des
beaux
arts,
Rouen.

11. Defects of mtDNA maintenance
Defects in replication machinery
• The replicative machinery (replisome) includes the catalytic
subunit of polymerase (encoded by the POLG gene), the
accessory subunit (encoded by POLG2), and the replicative
helicases Twinkle (encoded by PEO1) and DNA2
Defects involving the dNTP pool
• An adequate and balanced pool of the four dNTPs (dATP, dGTP,
dCTP and dTTP) is necessary to provide the precursors of
mtDNA replication
DiMauro S,, Schon EA, Carelli V, Hirano M. The clinical maze of
mitochondrial neurology Nat Rev Neurol. 2013.

12. Classification on the basis of functionally
distinct molecular defects:
Muta,ons
in
GENES
• Encoding
subunits
of
the
respiratory
chain
• Encoding
assembly
proteins
• Affec,ng
mtDNA
transla,on
• Controlling
the
phospholipid
composi,on
of
the
mitochondrial
inner
membrane
(MIM)
• Involved
in
mitochondrial
dynamics.
DiMauro S,, Schon EA, Carelli V, Hirano M. The clinical maze of
mitochondrial neurology Nat Rev Neurol. 2013.

13. Taylor RW, Turnbull DM. MITOCHONDRIAL DNA MUTATIONS IN HUMAN
DISEASE. Nature reviews Genetics. 2005;6(5):389-402. doi:10.1038/nrg1606.

15. Therapeutic Strategies
o Pre-implantation genetic diagnosis
o Gene therapy
– Vectors (viral or non viral) -mediated gene
transfer
• insertion of the corrective gene into an unpredictable
location within the chromosome : mutagenesis
• immunological response
o Mitochondrial replacement techniques
o Have raised questions on issues of safety and
ethics.

16. Therapeutic Strategies
Mitochondrial replacement techniques /
Cytoplasmic transfer
• For mtDNA-related diseases
• Nucleus of an oocyte from a carrier is transferred
to an enucleated oocyte from a normal donor
– the embryo will have the nDNA of the biological
parents but the mtDNA of a normal mitochondrial
donor.
– In human oocytes, cells were found to develop into
normal blastocysts and contain exclusively donor
mtDNA.

17. • Citoplasmatic transfer was done in the 90’s in USA for
unfertility problems
• Was banned in 2002 by FDA due to ethical and safety
concerns
– What is different from bone marrow transplant say critics, about
mitochondrial replacement, is that DNA from the donor will be
passed down future generations
Fig: The Human Fertilisation and Embryology Authority UK

18. Fig: The Human Fertilisation and Embryology
Authority UK

19. “could have uncontrollable and
unforeseeable consequences”
and would inevitably “affect the
human species as a whole”.
The House of Lords voted by 280 votes to 48
- March to August - The UK fertility
regulator will develop licensing
- Early Summer - The team in Newcastle
publish the final safety experiments
demanded by the regulator
- 29 October - Regulations come into
force
- 24 November - Clinics can apply to the
regulator for a licence
- By the end of 2015 - the first attempt
could take place

20. The hidden risks for ‘three-person’ babies
• Mismatch between nuclear and mtDNA
• Garry Hamilton : Nature 525, 444–446 (September 2015) doi:
10.1038/525444a
• Gretchen Vogel, Erik Stokstad: Science DOI: 10.1126/science.aaa7793
• Mitochondria Replacement can change
the expression profiles of nuclear genes

21. M Tachibana et al. Nature 000, 1-6 (2009) doi:10.1038/nature08368
Mito and Tracker, the first primates to be produced by
spindle-chromosomal complex transfer (ST) into enucleated
oocytes.
Mitochondrial gene replacement in primate offspring and
embryonic stem cells Nature 461, 367-372 (17 September 2009)
Alive and well at three years old
M. Tachibana, et al. Towards germline gene therapy of inherited
mitochondrial diseases Nature, 493 (2013), pp. 627–631

23. a polar body contains few mitochondria
and shares the same genomic material as
an oocyte, polar body transfer will prevent
the transmission of mtDNA variants

24. Delivery
of
recombinant
mtDNA
vectors
that
express
func,onal
replacement
copies
of
defec,ve
genes
from
within
the
mitochondrial
matrix.
Recoding
and
transloca,on
of
mitochondrial
genes
to
be
expressed
from
the
nucleus,
and
their
gene
products
subsequently
targeted
to
mitochondria.
Therapeutic Strategies

25. Therapeutic Strategies
Most promising strategy for genetic manipulation of
mtDNA is directed to inhibiting mutant mtDNA
replication and transcription.
Based in some characteristics of mtDNA
• mtDNA is present in multiple copies per cell
– somatic cells contain approximately 1,000 copies
– Oocytes ≈ 100,000 copies
– ≈ 1 to 10 copies in each mitochondrion.
P. Sutovsky, R.D. Moreno, J. Ramalho-Santos, T. Domiko, C. Simerly, G. Schatten
Ubiquitin tag for sperm mitocondria Nature, 403 (1999)

26. • mtDNA replicates continuously and
independently of cell division
• Cells with mtDNA mutations are
heteroplasmic
– containing different proportions of normal
and mutant mtDNA
– resulting from random segregation of
mtDNA during mitochondrial replication.
There
is
a
propor,on
of
mutated
mtDNA
necessary
for
the
disease
to
be
expressed
STRATEGY
IS
TO
INDUCE
A
SHIFT
IN
THIS
PROPORTION

27. Shifting of heteroplasmy
Lower the mutation load to subthreshold levels.
- Santra S, et al. Ketogenic treatment reduces deleted
mitochondrial DNAs in cultured human cells. Ann Neurol.
2004
- Clark et al. showed that necrosis of myopathic patient’s
skeletal muscle was followed by regeneration from
satellite cells without mutant mtDNA.
- Genetic approach:
- use of restriction endonucleases to eliminate specific
pathogenic mutations.
- GENOME EDITING

28. Heteroplasmic Shift
2. Importación mitocondrial
Nat. Genet., 15 (1997), pp. 212–215
Antigenomic PNA treatment for cells with A8344G MERRF
mutation
PNAs specifically inhibited replication of mutant but not wild-type
mtDNA templates

29. Genome editing starts with DNA double stranded cleavage
Nonhomologous end-
joining (NHEJ)
Homologous
recombination (HR)
Donor DNA
Figure adapted from : Hsu, Lander, Zhang: Development and Applications of CRISPR-Cas9 for Genome Engineering; Cell 2014
And
endogenous mechanisms of DNA repair
RESTRICTION
ENZYMES

30. Tanaka M, et al. Gene therapy for mitochondrial
disease by delivering restriction endonuclease SmaI
into mitochondria. J Biomed Sci. 2002
• T8993G mutation in mtDNA affects subunit 6 of
mitochondrial ATPase
– NARP (neuropathy, ataxia and retinitis pigmentosa)
– MILS (maternally-inherited Leigh syndrome)
• heteroplasmic conditions
• MILS usually has >90%mutant loads
• NARP usually associated with mutant loads of 60–90%.
– Mutant loads of less than 60% are generally
asymptomatic.

31. Tanaka M, et al. Gene therapy for mitochondrial disease by
delivering restriction endonuclease SmaI into mitochondria.
J Biomed Sci. 2002

32. This
destruc,on
proceeds
in
a
,me-­‐
and
dose-­‐dependent
manner
and
results
in
cells
with
significantly
increased
rates
of
oxygen
consumpCon
and
ATP
producCon.
Selec,ve
destruc,on
of
mutant
mtDNA.
Infec,on
with
an
adenovirus,
encoding
this
mitochondrially
targeted
R.XmaI
restric,on
endonuclease
T8993G
transversion
generates
a
unique
recogniCon
site
for
SmaI
and
XmaI
restric,on
endonucleases
(REs),
which
is
absent
in
wild
type
mtDNA

33. • T8993G
• Although more than 200 mutations in mt
DNA are asssociated with mt disease
• Only human mutation that generates a
unique restriction site that can be targeted
using the naturally occurring restriction
endonuclease smaI

34. Genome editing starts with DNA double stranded cleavage
Nonhomologous end-
joining (NHEJ)
Homologous
recombination (HR)
Donor DNA
ZFNs
CAS9:sgRNATALENs
HEs
Figure adapted from : Hsu, Lander, Zhang: Development and Applications of CRISPR-Cas9 for Genome Engineering; Cell 2014
And
endogenous mechanisms of DNA repair

35. There are currently four families of
engineered nucleases being used:
Zinc
finger
nucleases
(ZFNs),
Transcrip,on
Ac,vator-­‐Like
Effector
Nucleases
(TALENs),
CRISPR/Cas
system
Engineered
meganuclease-­‐engineered
homing
endonucleases.
(Cai and Yang, 2014; Gaj et al., 2013; Kim and Kim, 2014).

36. All these technics utilize
based
on
engineered
proteins
or
RNA
that
target,
and
specifically
bind,
to
a
designated
sequence
of
the
genome.
at
specific
loca,ons
double
stranded
breaks
(DSBs)
in
DNA
Restric,on
enzymes

37. ZINC FINGERS NUCLEASES
• Developed in early 2000s
Bibikova, M.,et als. Enhancing gene targeting with designed zinc finger nucleases.
Science 2003
Wolfe, S.A et als DNA recognition by Cys2His2 zinc finger proteins. Annu. Rev. Biophys.
Biomol. Struct. 2000
• The fusion of two components forms a ZFN:
– a sequence of 3 to 6 zinc finger proteins
• each zinc finger recognizes a DNA 3 base pair sequence
– the restriction enzyme FokI which only cleaves DNA when
it forms dimers
DNA template.
2.2. Transcription activator-like effector nucleases
Shortly after the discovery of ZFNs for specific genome editing, a
new class of DNA binding proteins was discovered in gram-negative
plant pathogens such as Xanthomonas termed transcription activa-
tor-like effectors (TALEs) (Fujikawa et al., 2006; Wright et al., 2014).
Each TAL effector protein contains 34 amino acids that were found
to be largely similar in composition except for two amino acids at
positions 12 and 13 (Boch et al., 2009; Moscou and Bogdanove,
2009). A total of four TAL effector proteins with specific domains
were found to bind each of the four individual amino acids guanine
(G), adenine (A), cytosine (C), and thymine (T), respectively along
the major groove of the DNA double helix. This 1:1 binding affinity
between TALEs and the four DNA bases allows for the construction
of a TALE array that can recognize any DNA sequence.
target any 20-bp DNA sequence (Mali et al., 2013). The following
details of the DNA recognition and subsequent double stranded
cleavage by CRISPR/Cas9 have been the subject of many reviews
(Doudna et al., 2014; Liu and Fan, 2014; Sander and Joung, 2014).
An illustration of the CRISPR/Cas9 system is shown in Fig.4.
The CRISPR/Cas9 system represents a departure from the
technologies of ZFNs and TALENs. The Cas9 endonuclease operates
as a monomer to induce DSBs, whereas the FokI in ZFNs and TALENs
operates as a dimer. The enzymatic machinery remains the same for
any intended target; only the guide RNA provides DNA binding
affinity and therefore targeting. Thus, CRISPR/Cas9 requires no
protein engineering foranychange in target,onlysynthesis of a new
guide RNA. This simplicity has dramatically reduced the time
needed to conduct genome engineering experiments.
3. Evidence of therapeutic potential
Each of the three technologies described above have spent
variable amounts of time being tested for therapeutic efficacy and
Fig. 2. An illustration of a zinc finger nuclease (ZFN) pair is shown. A ZFN consists of left and right monomers of typically 3 to 6 zinc finger proteins (ZFPs) and the FokI
restriction enzyme, which cleaves DNA when a dimer is formed. Each ZFP recognizes a target 3 base pair DNA sequence.
J.S. LaFountaine et al. / International Journal of Pharmaceutics 494 (2015)
180–194

39. ZFNs other uses
• Modify triplet repeats disorders .
• Generate double-strand breaks (DSBs) to
shrink CAG repeats to less toxic lengths
• Mittelman, D et als. Zinc-finger directed double-strand breaks within
CAG repeat tracts promote repeat instability in human cells. Proc
Natl Acad Sci U S A. 2009

40. TALENs èMito-TALENs
Transcription Activator-Like Effector Nucleases
Discovered in gram-negative plant pathogens such as Xanthomonas termed
transcription activator-like effectors (TALEs)
Fujikawa, T. et als , . Mol. Plant Microbe Interact 2006.
Wright, D.A., et als. TALEN-mediated genome editing: prospects and perspectives. Biochem. J. 2014
Bacman,
R.
et
als
Specific
elimina2on
of
mutant
mitochondrial
genomes
in
pa2ent-­‐derived
cells
by
mitoTALENs,”
Nature
Medicine,
2013.
• TALEN
has
been
reengineered
to
localize
to
mitochondria
and
specifically
remove
truncated
dysfunc,onal
mtDNAS
In
cybrid
cells-­‐
with
LHON
muta,on-­‐
complex
I
ac,vity
was
increased.
J.S. LaFountaine et al. / International Journal of Pharmaceutics 494 (2015)
180–194

41. ”The CRISPRs/Cas9 Revolution.
Came from studies of how bacteria fight infection
A CRISPR array is composed of a series of repeats interspaced by
spacer sequences acquired from invading genomes
This sequence is transcribed as crRNA which guides CRISPR-
associated (Cas) protein(s) to analogous invading genomes
introducing a DSB in the pathogenic DNA, inhibiting integration and
replication of the pathogen
Research tool and a cause for public concern.
turned out to be a system that can be programmed for binding and
cutting DNA.
Terns, M.P., Terns, R.M., 2011. CRISPR-based adaptive immune systems.
Curr. Opin. Microbiol. 14, 321–327.
Clustered Regularly Interspaced Short Palindromic Repeats

42. PALINDROMIC SEQUENCE
NON PALINDROMIC SEQUENCE
A
T
T
G
A
C
A
T
T
G
A
C
C
G
T
T
A
C
G
C
A
A
T
G
PALINDROMIC
Recogni,on
Sequence

44. Science 17 August 2012: Vol. 337 no. 6096 pp. 816-821
DOI: 10.1126/science.122582
Umeå University, Sweden.
University of California, Berkeley USA.
“We identify a DNA interference mechanism involving a dual-RNA structure that
directs a Cas9 endonuclease to introduce site-specific double-stranded breaks in
target DNA.”
“We propose an alternative methodology based on RNA-programmed Cas9 that
could offer considerable potential for gene-targeting and genome-editing
applications.”

45. Genome Editing
Cas9
endonuclease
operates
as
a
monomer
to
induce
DSBs
• FokI
in
ZFNs
and
TALENs
operates
as
a
dimer.
The
guide
RNA
(gRNA)
provides
the
targe,ng
DNA
.
CRISPR/Cas9
requires
no
protein
engineering
for
any
change
in
target,
only
synthesis
of
a
new
guide
RNA.
(gRNA)

46. • CRISPR/Cas9-mediated genome editing can be
successfully employed to manipulate the mitochondrial
genome.
• Still needs further study to understand how gRNA can be
translocated into the mitochondria matrix together with
mitochondria-localizing Cas9.

47. February, 2013
Multiple guide sequences can be encoded into a single CRISPR array to
enable simultaneous editing of several sites within the mammalian genome,
demonstrating easy programmability and wide applicability of the RNA-
guided nuclease technology.

48. Cas9 from Streptococcus pyogenes, known as spCas9, introducedas an RNA-
guided endonuclease by Charpentier and Doudna in 2012 has been the gold-
standard for CRISPR-based genome editing
Cell 163, 1–13, October 22, 2015 ª2015
Elsevier Inc.
Feng Zhang
Broad Institute of MIT
and Harvard, Cambridge

49. Shift Heteroplasmic
NZB/BALB heteroplasmic mice, which contain two mtDNA haplotypes, BALB and NZB
Selective Elimination of Mitochondrial Mutations in the Germline by Genome Editing Reddy,
Pradeep et al. Cell , 2015
C NZB
T BALB
- Specific elimination of BALB
mtDNA in NZB/BALB oocytes
and one-cell embryos.
- Prevented germline
transmission
- Using either mitochondria-
targeted restriction mito-ApaLi
or TALENS.

50. THANK YOU
MUCHAS GRACIAS