Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing

Human Molecular Genetics, 2009, Vol. 18, No. 17 doi:10.1093/hmg/ddp264 Advance Access published on June 4, 2009 3266–3273 Correction of tau mis-splicing caused by FTDP-17 MAPT mutations by spliceosome-mediated RNA trans-splicing Teresa Rodriguez-Martin1,2, Karen Anthony1, Mariano A. Garcia-Blanco3, S. Gary Mansfield4, Brian H. Anderton2 and Jean-Marc Gallo1, 1 MRC Centre for Neurodegeneration Research, Department of Clinical Neuroscience, King’s College London, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK, 2MRC Centre for Neurodegeneration Research, Department of Neuroscience, King’s College London, Institute of Psychiatry, De Crespigny Park, London SE5 8AF, UK, 3Center for RNA Biology, Department of Molecular Genetics and Microbiology, Duke University Medical Center, Durham, NC 27710, USA and 4VIRxSYS Inc., 200 Perry Parkway, Gaithersburg, MD 20877, USA Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by mutations in the MAPT gene, encoding the tau protein that accumulates in intraneuronal lesions in a number of neurodegenerative diseases. Several FTDP-17 mutations affect alternative splicing and result in excess exon 10 (E10) inclusion in tau mRNA. RNA reprogramming using spliceosome-mediated RNA trans-splicing (SMaRT) could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. SMaRT creates a hybrid mRNA through a trans-splicing reaction between an endogenous target pre-mRNA and a pretrans-splicing RNA molecule (PTM). However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. Excess E10 inclusion in FTDP-17 can be caused by intronic mutations destabilizing a stem-loop protecting the 50 splice site at the E10/intron 10 junction. COS cells transfected with a minigene containing the intronic 114 mutation produce exclusively E101 RNA. Generation of E102 RNA was restored after co-transfection with a PTM designed to exclude E10. Similar results were obtained with a target containing the exonic N279K mutation which strengthens a splicing enhancer within E10. Conversely, increase or decrease in E10 content was achieved by trans-splicing from a target carrying the D280K mutation, which weakens the same splicing enhancer. Thus E10 inclusion can be modulated by trans-splicing irrespective of the strength of the cis-splicing elements affected by FTDP-17 mutations. In conclusion, RNA trans-splicing could provide the basis of therapeutic strategies for impaired alternative splicing caused by pathogenic mutations in cis-acting splicing elements. INTRODUCTION Tauopathies are major diseases of the central nervous system characterized neuropathologically by intracellular filamentous inclusions formed by the microtubule-associated protein tau in affected neurons (1,2). Tauopathies include dementias such as Alzheimer’s disease and some forms of frontotemporal dementia (3). Definitive evidence for the pathogenic importance of tau was provided by the discovery of dominant mutations in the MAPT gene, the gene encoding tau, in the rare dementia, frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) (4– 6).  To whom correspondence should be addressed at: MRC Centre for Neurodegeneration Research, Department of Clinical Neuroscience, King’s College London Institute of Psychiatry, Box PO37, De Crespigny Park, London SE5 8AF, UK. Tel: þ44 207 848 0404; Fax: þ44 207 708 0017; Email: # 2009 The Author(s). This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. Received May 5, 2009; Revised May 5, 2009; Accepted June 1, 2009 Human Molecular Genetics, 2009, Vol. 18, No. 17 3267 The MAPT gene comprises 16 exons and alternative splicing of exons 2, 3 and 10 generates six isoforms in the brain. Exon 10 (E10) encodes the second of four imperfect 31– 32 amino-acid microtubule-binding repeats in the C-terminal half of the protein. Exclusion or inclusion of E10 generate tau isoforms with three (3R tau, E102) or four (4R tau, E10þ) microtubule-binding repeats, the latter having an increased affinity for microtubules. E10 is expressed only in adults and E10þ and E102 isoforms are expressed in approximately equal amounts in adult human brain. To date, more than thirty-five MAPT mutations have been associated with FTDP-17. Most missense mutations reduce the affinity of tau for microtubules (7), whereas silent or intronic mutations affect E10 splicing and can result in an up to 6-fold excess of tau mRNA containing E10 and in an elevated 4R/3R ratio (4,6,8,9). Elevated 4R/3R ratio is likely to have functional consequences, for example in the regulation of axonal transport (10). From a therapeutic perspective, correcting defective alternative splicing is best achieved by direct intervention at the RNA level. RNA can be reprogrammed by using spliceosomemediated RNA trans-splicing (SMaRTw) (11). SMaRT creates a hybrid mRNA through a trans-splicing reaction mediated by the spliceosome between the 50 splice site of an endogenous target pre-mRNA and the 30 splice site of an exogenously delivered pre-trans-splicing RNA molecule (PTM). Conversely, a PTM can be designed to carry the 50 splice site and trans-splice to the 30 splice site of a specific target (12). A typical application of SMaRT is the correction of loss-of-function mutations. We have shown previously that E102 to E10þ tau RNA conversion could be achieved using SMaRT (13). Thus, SMaRT could be a method of choice to correct aberrant E10 splicing resulting from FTDP-17 mutations. However, FTDP-17 mutations affect the strength of cis-splicing elements and could favor cis-splicing over trans-splicing. For instance, most intronic mutations are clustered at the exon 10-intron 10 junction and disrupt a stemloop structure protecting the 50 splice site (14,15). Furthermore, with the exception of the P301L/S mutations, mutations within E10 are located in cis-acting splicing regulatory elements and promote E10 retention (16,17). We therefore addressed the question of the potential effectiveness of SMaRT to correct aberrant E10 splicing caused by FTDP-17 mutations. Here we show that elevated or reduced E10 inclusion caused by intronic or exonic FTDP-17 mutations can be reversed by trans-splicing. Hence, RNA trans-splicing could provide the basis for the development of promising therapeutic strategies for impaired alternative splicing, especially in the context of neurological diseases. RESULTS Tau PTM constructs We have shown previously that E10 could be introduced in tau RNA by SMaRT (13). We have now designed a PTM, TauPTM9, to exclude E10. TauPTM9 comprises a binding domain hybridizing with the 30 end of intron 9 and huma (...truncated)