RESUMO
Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.
Assuntos
Proteínas de Ligação ao Ferro/genética , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos/genética , RNA de Cadeia Dupla/genética , RNA Mensageiro/genética , Expansão das Repetições de Trinucleotídeos , Adolescente , Adulto , Linhagem Celular , Criança , Feminino , Fibroblastos/metabolismo , Fibroblastos/patologia , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patologia , Ataxia de Friedreich/terapia , Regulação da Expressão Gênica , Terapia Genética/métodos , Humanos , Íntrons , Proteínas de Ligação ao Ferro/agonistas , Proteínas de Ligação ao Ferro/metabolismo , Masculino , Oligonucleotídeos/metabolismo , Oligonucleotídeos Antissenso/metabolismo , Cultura Primária de Células , RNA de Cadeia Dupla/metabolismo , RNA Mensageiro/agonistas , RNA Mensageiro/metabolismo , Triazóis/química , FrataxinaRESUMO
Antisense oligonucleotides (ASOs) are synthetic oligonucleotides that alter expression of disease-associated transcripts via Watson-Crick hybridization. ASOs that function through RNase H or the RNA-induced silencing complex (RISC) result in enzymatic degradation of target RNA. ASOs designed to sterically block access of proteins to the RNA modulate mRNA metabolism but do not typically cause degradation. Here, we rationally design steric blocking ASOs to promote mRNA reduction and characterize the terminating mechanism. Transfection of ASOs complementary to constitutive exons in STAT3 and Sod1 results in greater than 70% reduction of mRNA and protein. The ASOs promote aberrant exon skipping and generation of premature termination codon (PTC)-containing mRNAs. We inhibit the nonsense-mediated mRNA decay (NMD) pathway and show that the PTC-containing mRNAs are recognized by the UPF1 ATPase, cleaved by the SMG6 endonuclease and degraded by the XRN1 cytoplasmic exonuclease. NMD surveillance, however, does not entirely explain the mechanism of decreased STAT3 expression. In addition to exon skipping, ASO treatment causes intron retention and reduction of chromatin-associated STAT3 mRNA. The application of steric blocking ASOs to promote RNA degradation allows one to explore more nucleotide modifications than tolerated by RNase H or RISC-dependent ASOs, with the goal of improving ASO drug properties.