ABSTRACT
Dystrophia myotonica type 1 (DM1), the most common muscular dystrophy in adults, results from expansion of a CTG repeat in the 3'-untranslated region of the dystrophia myotonica protein kinase gene (DMPK). Correction of the mutant DMPK transcript is a potential therapeutic strategy in DM1. We investigated the efficacy of artificial trans-splicing molecules (ATMs) to target and correct DMPK transcripts. ATMs designed to target intron 14 of DMPK pre-mRNA transcripts were tested for their ability to trans-splice the transcripts of a DMPK mini-gene construct and the endogenous DMPK transcripts of human myosarcoma cells (CCL-136). On agarose gel electrophoresis analysis, six of eight ATMs showed trans-splicing efficacy when applied to DMPK mini-gene construct transcripts, of which three were able to trans-splice endogenous DMPK pre-mRNA transcripts in myosarcoma cells, with trans-splicing efficiency ranging from 1.81 to 7.41%. These findings confirm that artificial trans-splicing can repair DMPK pre-mRNA and provide proof-of-principle evidence for this approach as potential therapeutic strategy for DM1.
Subject(s)
Myotonic Dystrophy/genetics , Protein Serine-Threonine Kinases/genetics , RNA Precursors/genetics , Trans-Splicing/genetics , Base Sequence , Gene Targeting/methods , Humans , Molecular Sequence Data , Myosarcoma/enzymology , Myosarcoma/genetics , Myotonic Dystrophy/enzymology , Myotonin-Protein Kinase , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection , Tumor Cells, CulturedABSTRACT
Cobalt-activated acylase form-2 from human uterine myosarcoma, lung adenocarcinoma and the adjacent tissue was isolated, purified and characterized. It was found, that the enzyme from the malignant neoplasms differed both from the normal tissue and benign tumor acylase-2 in its Km value, temperature optimum and effect of some ions. Some abnormal properties (pH dependence, activation by cobalt and thermostability) were common for benign and malignant tumors. In lung tissue adjacent to adenocarcinoma, some deviations of acylase properties were observed, similar to that found in tumor, indicating that biochemical changes may be present also in tissues unaffected by the neoplastic growth by the histopathological criteria.
