Novel therapies: gene replacement

Replacing defective mitochondrial genes using AAV-mediated engineering

AAV-mediated gene therapy targeting the liver can be very effective in those mitochondrial diseases characterized by accumulation of toxic compounds, or by specific liver dysfunction. Examples of the first group are ethylmalonic encephalopathy, a lethal infantile condition caused by accumulation of toxic hydrogen sulphide, and mitochondrial neuro-gastro-encephalomyopathy (MNGIE), characterized by mitochondrial DNA damage caused by accumulation of mutagenic thymidine.

Figure | AAV8 therapy in Ethe1-/- mice | The AAV-treated Ethe1-/- mice (red line) have a highly prolonged lifespan compared to naive littermates (grey line).

In both cases, AAV-based liver-targeted gene replacement produced a persistent reduction of the toxic compound in body fluids and tissues of the corresponding disease mouse models. In particular, in the Ethe1 knockout mouse, which if untreated does not survive beyond P30, this effect was also associated with remarkable prolongation of lifespan, up to 8-12 months

Figure | AAV-mediated gene therapy | Several AAV-serotypes (AAV1, 2, 8, 6, 9) have been used to deliver and express several therapeutic genes (restriction enzymes, RE, ApaLI, AIFM1, TYMP, ETHE1, ND4, MPV17) to specific tissues.

We are now implementing a clinical trial using AAV-mediated gene therapy for MNGIE, for which we are members of a European consortium.

Other approaches

Other approaches are still at the experimental level. These include:

  • The use of alternative xenogenes encoding AOX and Ndi1 to bypass defects in complex III/IV and complex I, respectively. AOX is an alternative oxidase used by plants, several invertebrates, and lower eukaryotes to deliver electrons from coenzyme-Q to molecular oxygen, thus bypassing complexes III and IV; Ndi1 is used by several yeast species to oxidize NADH.

Figure | Electron-transport chain and the site of action of AOX

  • The modulation of mitochondrial morphology and/or dynamics. We have recently shown [bib][\bib] that moderate overexpression of Opa1, a GTPase of the inner mitochondrial membrane involved in shaping the mitochondrial cristae, leads to partial correction of the clinical and biochemical phenotype of two models of mitochondrial disease.


  • In collaboration with Prof. Giulia d’Amati, La Sapienza University, Rome, Italy we have observed that both reduced respiratory and proliferation rates of cybrids carrying the homoplasmic 3243G>A “MELAS” mutation in mt-tRNALeu(UUR) can be corrected by overexpressing the 60aa C-terminal peptide of the cognate aminoacyl-tRNA synthase, LARS2 (C-term), or (iv) a recombinant peptide resulting by the N-terminal fusion of C-term with a mitochondrial targeting sequence (MTS). These results suggest that the C-term peptide has a chaperone-like effect that stabilizes the mutant tRNA and can be exploited therapeutically. The recent availability of mouse models carrying mutations in mtDNA-encoded genes will allow us in the future to assess the efficacy of this approach in vivo.