Hereditary Spastic Paraplegia (HSP) is a heterogenous group of inherited disorders characterized by progressive spasticity and weakness of the lower limbs. HSP-50 or SPG50 is a rare subtype caused by biallelic pathogenic variants in AP4M1, encoding the M1 subunit of the heterotetrameric AP-4 complex, which is involved in protein recognition and sorting from the trans-golgi network to the endosomal-lysosomal system.
We identified a paternally inherited (c.1129del; p.Leu377PhefsTer67) and a maternally inherited (c.974+115dup) AP4M1 variant in a 4-year-old patient with progressive lower extremity spasticity. RT-PCR confirmed that the intronic duplication creates a novel splice site, causing an 112-base pair extension of exon 12. The pseudoexon insertion, shifts the reading frame, creating an early stop codon that is predicted to generate a truncated AP4M1 protein or undergo nonsense mediated mRNA decay. This variant is potentially therapeutically actionable by an antisense oligonucleotides approach to modulate gene expression and restore normal splicing. This is of particular interest, as haploinsufficiency of AP4M1 is not associated with disease.
Here, three 2’MOE ASO’s with phosphorothioate linkages were designed to block the AP4M1 novel splice site allowing splicing machinery to revert to the natural splice site and restore normal splicing. In multiple dose curve analysis on cultured patient fibroblasts, reverse transcription and digital PCR were used to determine the efficacy and accuracy of the ASO’s. One ASO was most effective at reducing pseudoexon transcript levels, without reducing total levels of AP4M1. Functional assays will be performed to determine the ASO’s effect on AP4M1 transport function. This study provides proof of concept for an exon skipping therapy for a specific HSP associated AP4M1 variant.