Nonsense suppressor therapies rescue peroxisome lipid metabolism and assembly in cells from patients with specific PEX gene mutations

Abstract

Peroxisome biogenesis disorders (PBDs) are multisystemic autosomal recessive disorders resulting from mutations in PEX genes required for normal peroxisome assembly and metabolic activities. Here, we evaluated the potential effectiveness of aminoglycoside G418 (geneticin) and PTC124 (ataluren) nonsense suppression therapies for the treatment of PBD patients with disease-causing nonsense mutations. PBD patient skin fibroblasts producing stable PEX2 or PEX12 nonsense transcripts and Chinese hamster ovary (CHO) cells with a Pex2 nonsense allele all showed dramatic improvements in peroxisomal very long chain fatty acid catabolism and plasmalogen biosynthesis in response to G418 treatments. Cell imaging assays provided complementary confirmatory evidence of improved peroxisome assembly in G418-treated patient fibroblasts. In contrast, we observed no appreciable rescue of peroxisome lipid metabolism or assembly for any patient fibroblast or CHO cell culture treated with various doses of PTC124. Additionally, PTC124 did not show measurable nonsense suppression in immunoblot assays that directly evaluated the read-through of PEX7 nonsense alleles found in PBD patients with rhizomelic chondrodysplasia punctata type 1 (RCDP1). Overall, our results support the continued development of safe and effective nonsense suppressor therapies that could benefit a significant subset of individuals with PBDs. Furthermore, we suggest that the described cell culture assay systems could be useful for evaluating and screening for novel nonsense suppressor therapies.

Therapeutics Based on Stop Codon Readthrough

“…Another study also questioned the ability of PTC124 to suppress PTCs because it was unable to mediate readthrough using multiple readthrough reporter constructs in cultured cells (85). Furthermore, PTC124 was unable to restore functional protein expressed from endogenous mutant transcripts in cell models associated with obesity (melanocortin 4 receptor) (14), peroxisome biogenesis disorders (30), and long-QT syndrome (53). …”

Nonsense-mediated decay in genetic disease: Friend or foe?

“…These compounds have been shown in both in vitro and in vivo models to alleviate disease pathogenesis by enhancing PTC read-through [119]. Examples include cystic fibrosis (CF) [120-131], Becker and Duchenne muscular dystrophy (BMD/DMD) [128, 129, 132-140], ataxia telangiectasia [139, 141, 142], Rett syndrome (RTT) [143-146], Usher syndrome type I (USH1) [147-149], Hurler syndrome (MPS1) [150-154], Maroteaux-Lamy syndrome (MPSVI) [155], carnitine palmitoyltransferase 1A (CPT1A) [156], hemophilia [157, 158], methylmalonic acidura (MMA) [159], neuronal ceroid lipofuscinosis (NCL) [114, 160, 161], spinal muscular atrophy (SMA) [162], peroxisome biogenesis disorder (PBD) [163, 164], obesity [165], poor drug metabolism [166], and cancer [151]. …”

Hepatic dysfunction in peroxisomal disorders

“…For PBD due to misfolded proteins, drugs belonging to the class of chaperones, are promising agents to restore peroxin function, as shown in fibroblasts [153]. To overrule premature stop mutations nonsense suppressor therapies have been tested in fibroblasts [154].…”