RESUMO
There is currently an urgent need for biomarkers that can be used to monitor the efficacy of experimental therapies for Duchenne Muscular Dystrophy (DMD) in clinical trials. Identification of novel protein biomarkers has been limited due to the massive complexity of the serum proteome and the presence of a small number of very highly abundant proteins. Here we have utilised an aptamer-based proteomics approach to profile 1,129 proteins in the serum of wild-type and mdx (dystrophin deficient) mice. The serum levels of 96 proteins were found to be significantly altered (P < 0.001, q < 0.01) in mdx mice. Additionally, systemic treatment with a peptide-antisense oligonucleotide conjugate designed to induce Dmd exon skipping and recover dystrophin protein expression caused many of the differentially abundant serum proteins to be restored towards wild-type levels. Results for five leading candidate protein biomarkers (Pgam1, Tnni3, Camk2b, Cycs and Adamts5) were validated by ELISA in the mouse samples. Furthermore, ADAMTS5 was found to be significantly elevated in human DMD patient serum. This study has identified multiple novel, therapy-responsive protein biomarkers in the serum of the mdx mouse with potential utility in DMD patients.
Assuntos
Proteínas ADAM/genética , Aptâmeros de Nucleotídeos/farmacologia , Biomarcadores Farmacológicos/sangue , Distrofina/genética , Distrofia Muscular de Duchenne/terapia , Oligonucleotídeos Antissenso/farmacologia , Proteínas ADAM/sangue , Proteína ADAMTS5 , Animais , Proteínas Sanguíneas/genética , Proteínas Sanguíneas/metabolismo , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/sangue , Proteína Quinase Tipo 2 Dependente de Cálcio-Calmodulina/genética , Modelos Animais de Doenças , Distrofina/agonistas , Distrofina/deficiência , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos mdx , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/metabolismo , Distrofia Muscular de Duchenne/patologia , Monoéster Fosfórico Hidrolases/sangue , Monoéster Fosfórico Hidrolases/genética , Proteínas Quinases/sangue , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases , Proteômica/métodosRESUMO
Antisense-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy. It aims to restore the dystrophin open reading frame by skipping exons with antisense oligonucleotides (AONs) to allow production of partly functional proteins. The approach is currently tested in phase 3 clinical trials, but dosing and maintenance regimens have not yet been well studied. This study compared pharmacokinetic and pharmacodynamic effects of different 2'-O-methyl phosphorothioate RNA AON dosing and maintenance regimens in the preclinical mdx mouse model. When comparing different dosing regimens over a period of 8 weeks, higher levels of AON, exon skipping, and protein were observed in muscle after low daily doses compared with large weekly doses. Secondly, after receiving a high loading dose (1,250 mg/kg) in the first week, mice treated with maintenance injections twice weekly for 8 weeks showed higher preservation of therapeutic effects than mice receiving less or no maintenance injections. In both cases, the regimen resulting in the highest AON and exon skipping levels in muscle also resulted in high AON levels in liver and kidneys. These studies underline the importance of balancing optimal AON efficacy and tolerable levels in non-target organs, which may be fine-tuned by further optimization of AON treatment regimens.