TAT-mediated Delivery of LAD Restores Pyruvate Dehydrogenase Complex Activity in the Mitochondria of Patients with LAD Deficiency  Matan Rapoport, Ann Saada, Orly Elpeleg, Haya Lorberboum-Galski  Molecular Therapy  Volume 16, Issue 4, Pages 691-697 (April 2008) DOI: 10.1038/mt.2008.4 Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 1 Schematic representation of TAT-LAD, TAT-Δ-LAD, and LAD fusion proteins, their purification, and in-vitro enzymatic activity. (a) Schematic representation of TAT-LAD fusion protein, and the control proteins—TAT-Δ-LAD (lacking the MTS moiety) and LAD (lacking the TAT moiety). (b) Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and Western blot using anti-LAD antibody analysis of purified TAT-LAD, TAT-Δ-LAD, and LAD fusion proteins. These proteins were purified using affinity chromatography as described in Materials and Methods. (c) Enzymatic activity of purified TAT-LAD, TAT-Δ-LAD, and LAD fusion proteins. The in vitro enzymatic activity assay was performed as described in Materials and Methods. The values presented are mean values ± SD of three separate enzymatic assays, each carried out in triplicate. LAD, lipoamide dehydrogenase; MTS, mitochondrial targeting sequence; TAT, transactivator of transcription peptide. Molecular Therapy 2008 16, 691-697DOI: (10.1038/mt.2008.4) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 2 Delivery of TAT-LAD into G229C/Y35X and E375K cells taken from patients. (a) Western blot analysis of whole-cell protein extracts from G229C/Y35X-treated cells using antibody against LAD. Both the TAT-LAD fusion protein (arrow) and the endogenous mutated LAD, corresponding to molecular weights of 58 and 50 kd, respectively, are observed. (b) LAD activity in treated G229C/Y35X cells. (c) LAD activity in E375K cells. Cells were treated with TAT-LAD, TAT-PAH, or LAD protein (0.075–0.1 μg/μl, final concentration) for different time periods. LAD activity was analyzed in whole-cell protein extracts, using the enzymatic activity assay described in Materials and Methods. Activity assays were conducted at least three times. The values presented are (b) the mean values ± SD or (c) depicting typical results. The enzymatic activity values are presented as nmol/min/mg protein. LAD, lipoamide dehydrogenase; PAH, phenylalanine hydroxylase; TAT, transactivator of transcription peptide. Molecular Therapy 2008 16, 691-697DOI: (10.1038/mt.2008.4) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 3 The fate of TAT-LAD and TAT-Δ-LAD within isolated mitochondria. (a) Radioactive-labeled TAT-LAD and the control protein TAT-Δ-LAD (lacking the mitochondrial targeting sequence) were expressed, in vitro and analyzed using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) autoradiography, matching their expected molecular sizes, 58 and 54 kd, respectively. (b) Mitochondria isolated from cells were incubated for 30 minutes with the radio-labeled proteins. The mitochondria were then washed, treated with proteinase K, and analyzed using SDS-PAGE autoradiography. The asterisk marks the 50 kd band of the processed TAT-LAD fusion protein. LAD, lipoamide dehydrogenase; TAT, transactivator of transcription peptide. Molecular Therapy 2008 16, 691-697DOI: (10.1038/mt.2008.4) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 4 Delivery of TAT-LAD into the mitochondria of G229C/Y35X cells taken from patients, and its effect on the LAD/CS ratio. The cells were treated with TAT-LAD or TAT-Δ-LAD (0.1 μg/μl, final concentration) for 4–6 hours. Subcellular fractions (cytosol and mitochondria) were separated in accordance with a differential centrifugation protocol as described in Materials and Methods. (a) Western blot analysis of the subcellular fractions showing the intracellular distribution of TAT-LAD and TAT-Δ-LAD, using anti-LAD antibody. The purity of the subcellular fractions was confirmed using the specific markers: E1α for the mitochondria and α-tubulin for the cytosol. (b) The LAD/CS ratio in the different mitochondrial fractions was calculated and normalized according to the relative in vitro enzymatic activity of each fusion protein respective to the basal LAD/CS ratio in the mitochondria of patients' fibroblasts. The results are presented in arbitrary units as the multiple of increase as compared to the basal LAD/CS ratio (1). CS, citrate synthase; LAD, lipoamide dehydrogenase; TAT, transactivator of transcription peptide. Molecular Therapy 2008 16, 691-697DOI: (10.1038/mt.2008.4) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions

Figure 5 PDHC co-localization and enzymatic activity in TAT-LAD-treated cells from patients. (a) D479V cells were treated with fluorescein isothiocyanate (FITC)-labeled TAT-LAD or LAD (green), washed, fixed, permeabilized, and incubated with anti-E1α antibody. The cells were then washed and incubated with anti-mouse Cy5 antibody (red). The cells were analyzed for co-localization using confocal microscopy (merge, yellow). Original magnifications: ×60 (LAD) and ×100 (TAT-LAD). (b–c) Cells were incubated with TAT-LAD (0.1 μg/μl, final concentration) for 3, 6, and 24 hours. PDHC activity assays were performed as described in Materials and Methods. (b) PDHC activity in treated E375K cells of patients. Activity values are presented as nmol/min/mg protein. (c) PDHC activity in treated E375K and D479V cells of patients. Activity values are presented as the percentage of normal PDHC activity measured in healthy fibroblasts in the same experiments. Activity assays were repeated three times. The values presented (in b and c) are the mean values ± SD. LAD, lipoamide dehydrogenase; PDHC, pyruvate dehydrogenase complex; TAT, transactivator of transcription peptide. Molecular Therapy 2008 16, 691-697DOI: (10.1038/mt.2008.4) Copyright © 2008 The American Society of Gene Therapy Terms and Conditions