C6ORF66 is an assembly factor of mitochondrial complex I.

Homozygosity mapping was performed in five patients from a consanguineous family who presented with infantile mitochondrial encephalomyopathy attributed to isolated NADH:ubiquinone oxidoreductase (complex I) deficiency. This resulted in the identification of a missense mutation in a conserved residue of the C6ORF66 gene, which encodes a 20.2 kDa mitochondrial protein. The mutation was also detected in a patient who presented with antenatal cardiomyopathy. In muscle of two patients, the levels of the C6ORF66 protein and of the fully assembled complex I were markedly reduced. Transfection of the patients' fibroblasts with wild-type C6ORF66 cDNA restored complex I activity. These data suggest that C6ORF66 is an assembly factor of complex I. Interestingly, the C6ORF66 gene product was previously shown to promote breast cancer cell invasiveness.

TAT-mediated delivery of LAD restores pyruvate dehydrogenase complex activity in the mitochondria of patients with LAD deficiency.

Modern medicine offers no cure for mitochondrial disorders such as lipoamide dehydrogenase (LAD) deficiency. LAD is the E3 subunit shared by alpha-ketoacid dehydrogenase complexes in the mitochondrial matrix, and these complexes are crucial for the metabolism of carbohydrates and amino acids. We propose a novel concept for restoring the activity of an immense multicomponent enzymatic complex by replacing one mutated component, the LAD subunit. Our approach entails the fusing of LAD with the transactivator of transcription (TAT) peptide, which is capable of rapidly crossing biological membranes, thereby allowing TAT-LAD to be delivered into cells and their mitochondria where it can replace the mutated endogenous enzyme. Our results show that TAT-LAD is indeed delivered into the cells and their mitochondria, where it is processed, restoring LAD activity to normal values and, most importantly, increasing the activity of pyruvate dehydrogenase complex. We report here, for the first time, that TAT-mediated replacement of one mutated component restores the activity of an essential mitochondrial multicomponent enzymatic complex in cells of patients with enzyme deficiencies. We believe that this approach involving TAT-mediated enzyme replacement therapy (ERT) can be applied to the treatment of LAD deficiency as well as to other mitochondrial and metabolic disorders.

Successful TAT-mediated enzyme replacement therapy in a mouse model of mitochondrial E3 deficiency.

Medicine today offers no cure for patients suffering from mitochondrial disorders, such as lipoamide dehydrogenase (LAD; also known as E3) deficiency, and treatment is limited to symptomatic care. LAD is one of the components of the α-ketoacid dehydrogenase complexes, which are mitochondrial multienzyme complexes crucial for the metabolism of carbohydrates and amino acids. Recently, we tested the therapeutic approach for treating mitochondrial disorders whereby the activity of multicomponent complexes in the mitochondria is restored by TAT-mediated enzyme replacement therapy (ERT). The LAD deficiency disease was used before as a proof-of-principle in vitro, in patients' cells, utilizing the TAT-LAD fusion protein. In this report, we present successful TAT-mediated ERT in an in vivo mouse model using E3-deficient mice. We demonstrate the delivery of TAT-LAD into E3-deficient mice tissues and that a single administration of TAT-LAD results in a significant increase in the enzymatic activity of the mitochondrial multienzyme complex pyruvate dehydrogenase complex within the liver, heart and, most importantly, the brain of TAT-LAD-treated E3-deficient mice. We believe that this TAT-mediated ERT approach could change the management of mitochondrial disorders and of other metabolic diseases in modern medicine.

TAT-based drug delivery system--new directions in protein delivery for new hopes?

There has been great progress in the use of TAT-based drug delivery systems for the delivery of different macromolecules into cells in vitro and in vivo, thus circumventing the bioavailability barrier that is a problem for so many drugs. There are many advantages to using this system, such as the ability to deliver these cargoes into all types of cells in culture and into all organs in vivo. This system can even deliver cargoes into the brain across the blood-brain barrier. In addition, the ability to target specific intracellular sub-localizations such as the nuclei, the mitochondria and lysosomes further expands the possibilities of this drug delivery system to the development of sub-cellular organelle-targeted therapy. The therapeutic applications seem almost unlimited, and the use of the TAT-based delivery system has extended from proteins to a large variety of cargoes such as oligonucleotides, imaging agents, low molecular mass drugs, nanoparticles, micelles and liposomes. In this review the most recent advances in the use of the TAT-based drug delivery system will be described, mainly discussing TAT-mediated protein delivery and the use of the TAT system for enzyme replacement therapy.