Antisense oligonucleotide treatment for a pseudoexon-generating mutation in theNPC1gene causing Niemann-Pick type C disease

Abstract

Niemann-Pick type C disease is an autosomal recessive disorder caused by mutations in either the NPC1 or NPC2 gene. While most of the mutations are missense, a few splicing mutations have also been described. We identified and characterized a novel point mutation c.1554-1009G>A located in intron 9 of the NPC1 gene in a Spanish patient. Sequencing of the cDNA from the patient showed that this intronic mutation creates a cryptic donor splice site resulting in the incorporation of 194 bp of intron 9 as a new exon (pseudoexon) in the mRNA. This new transcript bears a premature termination codon and is degraded by the nonsense-mediated mRNA decay mechanism. Experimental confirmation that the point mutation generates the inclusion of a pseudoexon in the mRNA was obtained using a minigene. A specific antisense morpholino oligonucleotide targeted to the cryptic splice site was designed and transfected into fibroblasts from the patient. Using this approach, normal splicing was restored. These results demonstrate the importance of screening deep intronic regions and support the efficacy of antisense therapeutics for the treatment of diseases caused by pseudoexon-generating mutations.

Niemann-Pick Type C disease: characterizing lipid levels in patients with variant lysosomal cholesterol storage

“…Although some of these cases may be explained by rearrangements in deep intronic or noncoding regulatory elements that are diffi cult to assess ( 5 ), the existence of one or more additional disease-causing genes cannot be excluded.…”

Present and future of antisense therapy for splicing modulation in inherited metabolic disease

“…Although most reported splicing mutations disrupt the conserved 3' and 5' splice sites at the exon-intron junctions, aberrant splicing may also be caused by mutations within introns that create or activate novel splice sites which are used in combination with opportunistic complementary sites, resulting in the inappropriate inclusion of intronic sequences, usually known as pseudoexons given their resemblance to true exons with potential 3' and 5' splice sites. Several examples of this pathogenic mechanism have been shown for diseases such as cystic fibrosis (OMIM 219700) (Friedman et al 1999), ataxia telangiectasia (OMIM 208900) (Du et al 2007), neurofibromatosis type 1 (OMIM 162200) (Pros et al 2009), and many different IMD, including organic acidemias (Rincon et al 2007;Tsuruta et al 1998), lysosomal disorders (Rodriguez-Pascau et al 2009;Vervoort et al 1998), congenital disorders of glycosylation (Schollen et al 2007), and tetrahydrobiopterin deficiencies (Ikeda et al 1997;Meili et al 2009). The frequency of this type of changes has been calculated in some diseases, ranging from 2-7% of the total alleles (Gurvich et al 2008;Pros et al 2009).…”

“…To date, there are many examples in the literature of the use of AO to exclude an intronic sequence (pseudoexon) activated by a point mutation in the final transcript ( Fig. 1a), including several IMD (Du et al 2007;Pros et al 2009;Rincon et al 2007;Rodriguez-Pascau et al 2009;Vega et al 2009) (Table 1). In addition, splicing intervention with AO has also been used to induce removal of in-frame exons containing a mutation (Fig.…”

Antisense Mediated Splicing Modulation For Inherited Metabolic Diseases: Challenges for Delivery

“…We showed that the use of a specific SSO targeted to the cryptic splice site activated by the mutation restored normal splicing in fibroblasts of the patient (Fig. 2) (Rodriguez-Pascau et al, 2009). This novel mutation (c.1554-1009G > A) was recently identified in another Spanish patient (Macias-Vidal et al, 2011) and in two twins from the United States (Chris Hempel, the Addi and Cassi Fund, personal communication), indicating that it is not a private mutation.…”