Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA A. Rincón, C. Aguado, L.R.

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Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA A. Rincón, C. Aguado, L.R. Desviat, R. Sánchez-Alcudia, M. Ugarte, B. Pérez The American Journal of Human Genetics Volume 81, Issue 6, Pages 1262-1270 (December 2007) DOI: 10.1086/522376 Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 1 Schematic representation of MUT, PCCA, and PCCB regions around the pseudoexons. Exons and pseudoexons are boxed. The inserted intronic sequence is shown in uppercase letters, and the surrounding intronic sequence is in lowercase letters. The sequence of the AMO used is underlined, and splice scores calculated with the BDGP software are denoted above the corresponding 5′ and 3′ splice sites. The mutations are denoted by arrows. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 2 Splicing assay for the wild-type and mutant minigenes corresponding to the MUT (A), PCCA (B), and PCCB (C) intronic changes. The results of the RT-PCR analysis using vector-specific primers are shown along with the schematic representation of the transcripts obtained, which were characterized by sequence analysis. V = vector exonic sequences. The solid line in panel C corresponds to a cryptic exon generated during the cloning process. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 3 Correction of aberrant splicing of MUT, PCCA, and PCCB genes by AMO targeted to the pseudoexon 5′ or 3′ splice sites. A, RT-PCR analysis of the MUT-mutated cell line of total RNA extracted from untreated cells (0 βM) and treated for different times with the specified amounts of AMO targeted to the 5′ cryptic splice site. The sense primer used is located at the junction of exons 10 and 11, and the reverse primer is located in exon 13. B, RT-PCR analysis of the PCCA-deficient cell line untreated or treated for 72 h with 10 or 20 βM of the corresponding AMO targeted to the 3′ splice site. C, RT-PCR analysis of the PCCB-deficient cell line untreated or treated for 72 h with 10 or 20 βM of AMO targeted to the 5′ splice site. Lane C, Control cell line. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 4 Time course of correctly spliced MUT mRNA stability in fibroblasts treated with AMO. Shown is RT-PCR analysis using primers to amplify MUT and GAPDH genes before treatment and after treatment for up to 25 d with 10 βM AMO targeted to the 5′ splice site. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 5 Functional correction of MCM activity after AMO treatment. MCM activity was measured by incorporation of [14C] into trichloroacetic acid–precipitable material in the control cell line (C) and the MUT-mutated cell line untreated or treated with 10, 15, or 20 βM of AMO targeted to the 5′ cryptic splice site. The cells were harvested 48 h after transfection. The data show the mean±SD from at least four independent experiments, and control data were obtained from four independent cell lines. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 6 Functional correction of PCC activity after AMO treatment. PCC activity was measured 72 h after transfection with 0, 10, or 20 βM of the corresponding AMO in PCCA- and PCCB-deficient cell lines. The black bar corresponds to control PCC activity measured in four independent cell lines. Dark-gray bars show PCC activity in PCCA-deficient fibroblasts treated with AMO targeted to the 3′ cryptic splice site. Light-gray bars show PCC activity in PCCB-deficient fibroblasts treated with AMO targeted to the 5′ cryptic splice site. The data show the mean±SD from at least three independent experiments. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions

Figure 7 Recovery of biotinylated PCCA protein after AMO treatment. Biotinylated proteins were detected in total cellular extract by avidin-alkaline phospahatase assay in the PCCA-deficient cell line untreated (lane 1) or 72 h after treatment with the corresponding AMO (lane 2). Lane 3, Hepatoma cellular extract. Shown are the biotin-containing α-subunits of methylcrotonylCoA carboxylase (MCCA) and PCCA. The American Journal of Human Genetics 2007 81, 1262-1270DOI: (10.1086/522376) Copyright © 2007 The American Society of Human Genetics Terms and Conditions