Targeting of RNA into mitochondria: mechanistic and

therapeutic insights

I. Tarassov Journée de la SFEIM 17 Novenmbre 2014, Paris, France

Energy Metabolism

Apoptosis Aging Biosyntheses

Defects in mitochondrial functions are often associated with myopathies and

neuropathies and play a critical role in many age-related diseases

Ophtalmoplegia

(decreased eye associated

Muscles tone)

Parkinson

Alzheimer

Cancer Vascular and heart diseases

In humans:

mtDNA 13 proteins, 2 rRNAs, 21 tRNAs

nDNA >1000 proteins, several ncRNAs (tRNAs, MRP RNA, Rnase P RNA, 5S rRNA)

Genetic double-dependence

Import of RNA into mitochondria

Yeast

Plants

Protozoans

Mammals

tRNAs

RNase P RNA

RNase MRP RNA

5S rRNA Mammals

Import determinants in yeast tRNA(Lys):

Alternative structure of RNA

Kolesnikova et al. (Science, 2000)

Entelis et al . (J Biol Chem, 2001)

Kolesnikova et al. (Mitochondrion, 2002)

Kolesnikova et al. (RNA, 2010)

Also Imported in an

artificial way in human

mitochondria

Smirnov et al. (RNA, 2008)

5S rRNA import determinants

Diabetes Thyroid

Disease Myopathy

Peripheral

Neuropathy

Deafness

CVA / Seizures /

Developmental delay

Respiratory Failure Optic Atrophy / Retinitis Pigmentosa

Cardiomyopathy

Short Stature

Marrow Failure

Liver

Failure

Neurological Non-Neurological

mtDNA mutations cause mitochondrial dysfunctions

>60

(% mutated mtDNA)

Cyto

ch

rom

e c

o

xid

ase a

cti

vit

y

100%

a b

*

*

*

*

*

*

*

*

*

*

mtDNA heteroplasmy and pathogenic threshold

Expression of site-specific

endonucleases

Inhibition of mutant mtDNA

replication

Miothochondrial diesease:

Possible gene therapy aproaches

« Allotopic » expression of

proteins or RNAs

Modulation of heteroplasmy

levels

Mito-TALENs (2013)

Mito-Zn-finger nucleases (2014)

Towards the model of gene therapy : MERRF

(Myoclonic Epilepsy and Red Ragged Fibers)

Which are the biochemical effects of the mutation ?

Hypomodification of the U34 (Yakuzawa et al., 2000)

Poor recognition of AAG codons on mRNA (Yakuzawa et al., 2001; Kirino et al. 2004, 2005)

Low complexes I (ND) & IV (Cox) activity

Low respirtation levels, low DY

Complementation of the MERRF mutation

Complementation of the MERRF mutation in human cells

tRK3 tRK2-93

Complementation is due to induced tRNA import

Expression and import of tRNAs in cybrid cells

Partial correction:

Respiration

DY

Translation

Complexes

I/IV

Cox-cytochemistry

Kolesnikova et al., Science 2000

Kolesnikova et al., Hum Mol Genet 2004

MELAS:

Mitochondrial Encephalomyopathy with Lactic Acidosis and Stroke-like

episodes

MELAS m.3243A>G mutation:

80% of MELAS cases

Carrier frequency ~1/10 000

Defect of Complex I (42% of patients),

Complex IV (29%), Complex III (23%)

Heteroplasmic

Mean onset ~ 10 years

Mean life span

~ 20 – 40 years

No treatement available

G

Recombinant tRNAs design

H. sapiens

mitochondrial

tRNALeu(UUR)

Kirino et al, 2005

CAA

G1-C72

5’

A

A73 A73

A

G C

tRK3

UAA UAA

CAA CAA

identity elements for hmt

LeuRS

determinants of tRNA

import into mitochondria

mutations introduced in

anticodon

Effect of tRK1CAA import on mitochondrial protein synthesis

1

0.8

0.6

0.4

0.2

0

Mel-pLKO Mel-T1CAA 143B

2.5

2

1.5

1

0.5

0

3

tRK1CAA import is acompanied by a generalized increase of mito-translation

Karicheva et al. (Nucleic Acids Res 2011)

Anti-replicative strategy: effect of antigenomic RNAs on heteroplasmy in cybrid cells

containing a deletion in mtDNA

Comte et al., Nucleic Acids Res 2013

Recombinant RNAs imported into

mitochondria of human cells in culture

caused a decrease of mutant mtDNA level.

Oligonucleotides corresponding to the target sequence were inserted into RNA vector molecules.

Stable expression of 5S rRNA-based recombinant RNA genes

intoduced into genome of cybrid cells harboring 65% of KSS

mtDNA molecules

Clones with decreased heteroplasmy levels

5S rRNA can be used as a vector for delivering RNA sequences into

human mitochondria

Oligonucleotides corresponding to the both strands of

target sequence were used to substitute the Loop C –

Helix III region of 5S rRNA

Comte et al., Nucleic Acids Res 2013

Mutation A13514>G (D393G) in ND5 gene, coding for respiratory Complex I subunit; MELAS like syndrome

and MELAS/Leigh/Leber hereditary optic neuropathy syndrome

Recombinant RNA (R20) caused a stable decrease of mutant

mtDNA level.

Tonin et al., J Biol Chem 2014

Developing the antigenomic strategy for point mutations in mtDNA

Design of RNA-vector molecules importable into human mitochondria:

optimisation of RNA stability

The new set of molecules importable into human mitochondria containing various

modifications.

Tonin et al., Biochimie 2014

Patent US 62/011,755 (2014)

Conclusions

In both allotopic and antigenomic strategies, the approach has a limited effect: levels of expression, aminoacylation (tRNA), import, accessibility to the target (antigenomic), etc. might be improved

Artificially established import of cytosolic-type tRNA into mitochondria can be used to deliver tRNAs with either normal or switched identities in human mitochondria to functionnally replace mutant tRNAs

Mitochondrially imported RNA molecules bearing oligonucleotide sequences complementary to mtDNA can affect in a specific manner mtDNA replication thus provoking induced changes of heteroplasmy

Small non-coding RNA molecules (tRNAs, tRNA-derivatives, 5S rRNA, etc.) can serve as vectors to deliver short oligoribonucleotides into mitochondria

Participants & sponsors

Team « Mito »

A.-M. Heckel N. Entelis B. Masquida A. Smirnov A. Smirnova R. Loutre I. Dovydenko I. Chicherin M. Baleva I. Tarassov

Former members:

R. Martin C. Comte O. Kolesnikova O. Karicheva Y. Tonin T. Schirtz M. Vyssokikh T. Schirtz A. Gowher

Collaborations:

P. Kamensky (Moscow University) A. Lombès (Hôpital Salpétrière, Paris) A. Rötig & A. Munnich (Hôpital Necker, Paris) A. Venyaminova (Inst. Fund. Med, Novossibirsk) O. Elpeleg (Jerusalem Univ.) R. N. Lightowlers (Newcastle Univ.) Funding: