Proceedings of The Physiological Society
King's College London (2011) Proc Physiol Soc 22, C12
Reprogramming of Tau isoforms by RNA trans-splicing: a potential gene therapy approach for tauopathies.
M. E. Avale1, T. Rodríguez-Martín1, J. M. Gallo1
1. MRC Centre for Neurodegeneration Research, Insitute of Psychiatry, King's College London, London, United Kingdom.
Tau (microtubule-associated protein Tau, MAPT) is a protein predominantly expressed in neuronal axons, which promotes microtubule polymerisation and stabilisation. The exon 10 (E10) of the MAPT gene encodes the second of four imperfect microtubule-binding repeats in the Tau protein. Exclusion or inclusion of E10 by alternative splicing gives rise to Tau isoforms with three (3R) or four (4R) microtubule-binding repeats, respectively. In the normal adult human brain the ratio 4R/3R is 1. Tauopathies are a group of neurodegenerative diseases characterized by the intracellular accumulation of Tau. Recent evidence suggests that some of these conditions are associated with abnormal alternative splicing at the level of E10, which affects the normal ratio between 4R/3R Tau isoforms. The aim of this project is to modulate the ratio between 4R/3R Tau isoforms by reprogramming the inclusion of Tau E10. The strategy used is the spliceosome-mediated RNA trans-splicing (SMaRT). SMaRT creates a chimaeric mRNA through a trans-splicing reaction between the 5’ splice site of an endogenous target pre-mRNA and the 3’ splice site of an exogenously delivered RNA molecule. To test this strategy in a prototypic model of Tau pathology, we used a mouse transgenic model that carries the human Tau gene in the mouse Tau knock out background (hTau mice). Trans-splicing molecules (PTMs) were delivered by lentiviral vectors into differentiated primary neurons of hTau mice. Different lentiviral concentrations (multiplicity of infection, moi) were used to achieve the best transduction efficiency. At moi=10 (i.e. 10 viral particles per neuron) PTMs produced efficient trans-splicing with the Tau RNA. The trans-spliced product was detected at the RNA level by RT-PCR and at the protein level by immunoprecipitation and western blot. The efficiency of Tau isoform conversion was on average 35 %. These results provide promising perspectives of a plausible gene therapy approach to correct aberrant RNA splicing in tauopathies.
Where applicable, experiments conform with Society ethical requirements