Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2007 Dec;81(6):1262-70.
doi: 10.1086/522376.

Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

Affiliations
Free PMC article

Propionic and Methylmalonic Acidemia: Antisense Therapeutics for Intronic Variations Causing Aberrantly Spliced Messenger RNA

A Rincón et al. Am J Hum Genet. .
Free PMC article

Abstract

We describe the use of antisense morpholino oligonucleotides (AMOs) to restore normal splicing caused by intronic molecular defects identified in methylmalonic acidemia (MMA) and propionic acidemia (PA). The three new point mutations described in deep intronic regions increase the splicing scores of pseudoexons or generate consensus binding motifs for splicing factors, such as SRp40, which favor the intronic inclusions in MUT (r.1957ins76), PCCA (r.1284ins84), or PCCB (r.654ins72) messenger RNAs (mRNAs). Experimental confirmation that these changes are pathogenic and cause the activation of the pseudoexons was obtained by use of minigenes. AMOs were targeted to the 5? or 3? cryptic splice sites to block access of the splicing machinery to the pseudoexonic regions in the pre-mRNA. Using this antisense therapeutics, we have obtained correctly spliced mRNA that was effectively translated, and propionyl coenzyme A (CoA) carboxylase (PCC) or methylmalonylCoA mutase (MCM) activities were rescued in patients' fibroblasts. The effect of AMOs was sequence and dose dependent. In the affected patient with MUT mutation, close to 100% of MCM activity, measured by incorporation of (14)C-propionate, was obtained after 48 h, and correctly spliced MUT mRNA was still detected 15 d after treatment. In the PCCA-mutated and PCCB-mutated cell lines, 100% of PCC activity was measured after 72 h of AMO delivery, and the presence of biotinylated PCCA protein was detected by western blot in treated PCCA-deficient cells. Our results demonstrate that the aberrant inclusions of the intronic sequences are disease-causing mutations in these patients. These findings provide a new therapeutic strategy in these genetic disorders, potentially applicable to a large number of cases with deep intronic changes that, at the moment, remain undetected by standard mutation-detection techniques.

Figures

Figure  1.
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.
Figure  2.
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.
Figure  3.
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 μM) 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 μM 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 μM of AMO targeted to the 5′ splice site. Lane C, Control cell line.
Figure  4.
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 μM AMO targeted to the 5′ splice site.
Figure  5.
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 μM 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.
Figure  6.
Figure 6.
Functional correction of PCC activity after AMO treatment. PCC activity was measured 72 h after transfection with 0, 10, or 20 μM 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.
Figure  7.
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.

Similar articles

See all similar articles

Cited by 25 articles

See all "Cited by" articles

Publication types

MeSH terms

Supplementary concepts

LinkOut - more resources

Feedback