Replacement of the C6ORF66 Assembly Factor (NDUFAF4) Restores Complex I Activity in Patient Cells

• Main Authors: Dana Marcus (Author), Michal Lichtenstein (Author), Ann Saada (Author), Haya Lorberboum-Galski (Author)
• Format: Book
• Published: BMC, 2013-05-01T00:00:00Z.
• Subjects: article
• Online Access: Connect to this object online.
• Notes: 10.2119/molmed.2012.00343; 1076-1551; 1528-3658
• Keywords: Chromosome 9 Open Reading Frame 72 (C6ORF66); Patient Cells; Assembly Factor; Protein Replacement Therapy; Gene Fusion Techniques; Therapeutics. Pharmacology; RM1-950; Biochemistry; QD415-436
• Source: Molecular Medicine, Vol 19, Iss 1, Pp 124-134 (2013)

Abstract Disorders of the oxidative phosphorylation (OXPHOS) system frequently result in a severe multisystem disease with the consequence of early childhood death. Among these disorders, isolated complex I deficiency is the most frequently diagnosed, accounting for one-third of all cases of respiratory chain deficiency. We chose to focus on complex I deficiency, caused by mutation in the assembly factor chromosome 6, open reading frame 66 (C6ORF66; NADH dehydrogenase [ubiquinone] complex I assembly factor 4 [NDUFAF4]) protein. We used the approach of cell- and organelle-directed protein/enzyme replacement therapy, with the transactivator of transcription (TAT) peptide as the moiety delivery system. This step will enable us to deliver the wild-type assembly factor C6ORF66 into patient cells and their mitochondria, leading to the proper assembly and function of complex I and, as a result, to a functional OXPHOS system. We designed and constructed the TAT-ORF fusion protein by gene fusion techniques, expressed the protein in an Escherichia coli expression system and highly purified it. Our results indicate that TAT-ORF enters patients' cells and their mitochondria rapidly and efficiently. TAT-ORF is biologically active and led to an increase in complex I activity. TAT-ORF also increased the number of patient cells and improved the activity of their mitochondria. Moreover, we observed an increase in ATP production, a decrease in the content of mitochondria and a decrease in the level of reactive oxygen species. Our results suggest that this approach of protein replacement therapy for the treatment of mitochondrial disorders is a promising one.