Abstract
Purpose :
Many retinal diseases, such as Age-Related Macular Degeneration and Macular Telangiectasia, may have compromised mitochondria and dysfunctional mitophagy. Gene therapy has the potential to treat such diseases but efficient mitochondrial targeting remains a challenge in the field. Our goal is to design and optimize AAV mediated gene therapy vectors for targeting mitochondria in photoreceptors and Müller glia. We aim to determine whether a nuclear-encoded transgene redirected to the mitochondria or a mito-targeted AAV capsid encoding a mitochondrial transgene is the more selective and/or efficient approach.
Methods :
We used computational methods to predict cleavable N-terminal mitochondrial targeting signal (MTS) pre-sequences that redirect selectively their target gene from the nucleus to the mitochondria. We cloned MTS sequences belonging to several families of mitochondrial genes at the N-terminal of tdTomato, to create fusion fluorescent transgenes. HEK293T and 661W cells were transfected with the different MTS-tdTomato constructs. Immunochemistry, qRT-PCR and western blots were performed to assess the efficiency/selectively of each MTS. Then, we engineered our 2 different systems of mito-targeting. 1) A nuclear-encoded MTS-tdTomato under the control of a CAG promoter (scCAG.MTS-tdTomato) was packaged with wild type 7m8 and ShH10. 2) A mitochondrial encoded tdTomato under the control a mitochondrial promoter (HSP) was packaged into mito-targeted 7m8 and ShH10 (MTS-VP2 supplied in trans). The rAAV constructs (mtAAV-HSP-tdTomato, AAV-MTS-tdTomato and AAV-tdTomato) were injected intravitreally into C57BL/6 mice. Animals were euthanized 3 weeks post-injections and retinas were collected for analysis.
Results :
The MTS sequence of the Ornithine Transcarbamylase (OTC) gene was the most selective at redirecting transgenes to the mitochondria in vitro. Other MTS sequences were also very efficient but less selective than OTC as they exhibit some cytoplasmic fluorescence as well. Our in vivo preliminary results also show that OTC is efficient at redirecting its transgene to the mitochondria. Further characterization is still needed to assess which of the mito-AAV or AAV-MTS methods is better at targeting the mitochondria in the outer retina and Müller glia.
Conclusions :
Based on these results we will be able to design better gene therapies to treat animal models of mitochondrial disease.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.