Engineering a tissue-specific promoter for the production of myosin expressing adenovirus and testing specificity and sensitivity of the RP10 siRNA for allele-specific silencing of the R1500P mutant in Laing distal myopathy.

Lanting, B.O. (Bart Oane) (2014) Engineering a tissue-specific promoter for the production of myosin expressing adenovirus and testing specificity and sensitivity of the RP10 siRNA for allele-specific silencing of the R1500P mutant in Laing distal myopathy. thesis, Medicine.

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Abstract

Introduction: The cellular and molecular phenotype of the R1500P mutant β-myosin gene, known to cause Laing distal myopathy, has recently been published. siRNA based therapy is a method for gene specific silencing through RNAi (RNA interference), thereby preventing the expression of protein. One strategy for studying myosin is to transduce cells using an adenoviral expression system. However, myosin appears to be detrimental to virus yield. We hypothesize that using a differentiated muscle cell-specific promoter will increase the production of myosin-expressing adenovirus and that blocking mutant protein expression by siRNA-based therapy will ameliorate Laing distal myopathy in vitro. Methods: NRVMs (neonatal rat ventricular myocytes) were co-transfected with a reaction mixture containing either two expression plasmids encoding mCherry tagged WT (wild type) myosin and GFP tagged mutant myosin, or two expression plasmids encoding mCherry tagged WT myosin and GFP tagged mutant myosin and the RP10 siRNA, which has been shown to successfully silence the R1500P mutation in COS7 cells, and subsequently visualized by confocal microscopy. Furthermore, the rat α-myosin heavy chain promoter region from -401 to -38 was tested for tissue-specificity compared to the CMV(cytomegalovirus)-promoter by transfection of NRVMs and HEK293 cells through measurement of luciferase activity. Results: The expression of the CMV- and α-promoter in HEK293 cells was 1654 and 5 RLU respectively. In NRVMs, CMV- and α-promoter expression was 1676 and 285 RLU respectively. Furthermore, the RP10 siRNA silencing of the R1500P myosin mutant expression was 6-fold compared to WT. Conclusion: We showed that the α-promoter demonstrated tissue-specific expression in cardiac cells as compared to the non-tissue-specific CMV-promoter, thereby paving the way for amplification of myosin expressing adenovirus. We also showed that the RP10 siRNA was successful in silencing the R1500P myosin mutant in NRVMs without appreciable effecting WT myosin expression, indicating the amelioration of Laing distal myopathy in vitro.

Item Type:	Thesis (Thesis)
Supervisor name:	Boer, Prof. Dr. RA de
Supervisor name:	Leinwand, Prof. Dr. LA and University of Colorado at Boulder, CO
Faculty:	Medical Sciences
Date Deposited:	25 Jun 2020 10:51
Last Modified:	25 Jun 2020 10:51
URI:	https://umcg.studenttheses.ub.rug.nl/id/eprint/1213

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