Therapeutic strategies based on modified U1 snRNAs and chaperones for Sanfilippo C splicing mutations

Abstract

BackgroundMutations affecting RNA splicing represent more than 20% of the mutant alleles in Sanfilippo syndrome type C, a rare lysosomal storage disorder that causes severe neurodegeneration. Many of these mutations are localized in the conserved donor or acceptor splice sites, while few are found in the nearby nucleotides.MethodsIn this study we tested several therapeutic approaches specifically designed for different splicing mutations depending on how the mutations affect mRNA processing. For three mutations that affect the donor site (c.234 + 1G > A, c.633 + 1G > A and c.1542 + 4dupA), different modified U1 snRNAs recognizing the mutated donor sites, have been developed in an attempt to rescue the normal splicing process. For another mutation that affects an acceptor splice site (c.372-2A > G) and gives rise to a protein lacking four amino acids, a competitive inhibitor of the HGSNAT protein, glucosamine, was tested as a pharmacological chaperone to correct the aberrant folding and to restore the normal trafficking of the protein to the lysosome.ResultsPartial correction of c.234 + 1G > A mutation was achieved with a modified U1 snRNA that completely matches the splice donor site suggesting that these molecules may have a therapeutic potential for some splicing mutations. Furthermore, the importance of the splice site sequence context is highlighted as a key factor in the success of this type of therapy. Additionally, glucosamine treatment resulted in an increase in the enzymatic activity, indicating a partial recovery of the correct folding.ConclusionsWe have assayed two therapeutic strategies for different splicing mutations with promising results for the future applications.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-014-0180-y) contains supplementary material, which is available to authorized users.

Molecular characterization of a large group of Mucopolysaccharidosis type IIIC patients reveals the evolutionary history of the disease

“…The frequency and worldwide distribution of the HGSNAT mutations in 25 new MPSIIIC index patients together with those previously reported (Ali Pervaiz et al, ; Canals et al, ; Coutinho et al, ; Fan et al, , ; Fedele & Hopwood, ; Feldhammer, Durand, Mrazova, et al, ; Hrebicek et al, ; Hu et al, ; Huh et al, ; Matos et al, ; Ouesleti et al, , ; Ruijter et al, ; Velasco et al, ) are summarized in Figure . The data show that Europe, South America, and Western Asia are so far the regions with the highest number of families reported with molecular diagnosis of MPSIIIC.…”

“…This late‐onset/slowly progressing group also included patients affected with different types of mutations: splicing (Can1 c.494−1G>A/c.494−1G>A; Pt1, c.234+1G>A/c.372‐2A>G), missense (Irn1, p.Pro283Leu/p.Pro283Leu; Trk2, p.Arg344Cys/p.Arg344Cys; USA1, p.Gly173Asp/p.Gly423Trp) or their combination (USA2, c.1250+1G>A/p.Ala489Glu). The effects of some of these mutations have been previously studied and several of them including Pro283Leu, Arg344Cys, and c.372‐2A>G were partially amenable by the active site‐specific chaperone glucosamine (Feldhammer, Durand, & Pshezhetsky, ; Matos et al, ). Thus, even a very small amount of the residual HGSNAT activity can ameliorate the clinical phenotype of MPSIIIC patients suggesting that for those affected with amenable mutations, chaperone therapy can become a feasible treatment option.…”

“…Mutations in the HGSNAT gene have been identified in MPSIIIC patients from several countries in Europe (Belarus, Belgium, Czech Republic, Finland, Germany, Greece, Ireland, Italy, Poland, Portugal, Spain, the Netherlands, and the UK), North Africa (Morocco and Tunisia), Asia (Turkey, Pakistan, Singapore, and South Korea), North (Canada and USA; Ali Pervaiz et al, ; Canals et al, ; Coutinho et al, ; Fan et al, ; Fan, Tkachyova, Sinha, Rigat, & Mahuran, ; Fedele & Hopwood, ; Fedele et al, ; Feldhammer, Durand, Mrazova, et al, ; Hrebicek et al, ; Hu et al, ; Huh et al, ; Matos et al, ; Ouesleti et al, , ; Ruijter et al, ) and South America (Argentina and Colombia; Canals et al, ; Velasco et al, ). Approximately 30% of all MPSIIIC variants are shared between different populations suggesting the existence of a common ancestor.…”

An Exon-Specific U1snRNA Induces a Robust Factor IX Activity in Mice Expressing Multiple Human FIX Splicing Mutants

“…Differently, our and other groups extensively exploited the physiological role of the U1snRNA to promote exon inclusion in the presence of exon-skipping mutations, 5,6,7,8,9,10,11,12,13,14,15,16,17 a relevant cause of severe forms of human genetic disease. 18,19,20 The first generation of engineered U1snRNA had a modified 5′ tail with increased complementarity to defective 5′ss, and were shown to rescue exon inclusion in several cellular 5,6,7,8,9,10,11,12,13,14,15,16,21 and also in vivo 17 disease models.…”

“…18,19,20 The first generation of engineered U1snRNA had a modified 5′ tail with increased complementarity to defective 5′ss, and were shown to rescue exon inclusion in several cellular 5,6,7,8,9,10,11,12,13,14,15,16,21 and also in vivo 17 disease models. However, these U1snRNAs are often tailored on the disease-causing mutation and have the intrinsic risk of off-target effects by recognizing the partially conserved donor splice site 22 in other splicing units.…”