Therapeutic application of allele-specific silencing by siRNA for gene duplication disorders: a proof-of-principle in Autosomal Dominant LeukoDystrophy (ADLD)

Anno:

2019

Therapeutic application of allele-specific silencing by siRNA for gene duplication disorders: a proof-of-principle in Autosomal Dominant LeukoDystrophy (ADLD)

Giorgio E1, Lorenzati M2, Rivetti di Val Cervo P3, Brussino A1, Cernigoj M3, Della Sala E1, Bartoletti Stella A4, Ferrero M1, Caiazzo M5,6, Capellari S4,7, Cortelli P4,7, Conti L8, Cattaneo E3,9, Buffo A2, Brusco A1,10.

2019 Jul 1;142(7):1905-1920. doi: 10.1093/brain/awz139.

Author information

1: University of Torino, Department of Medical Sciences, Torino, Italy.
2: University of Torino, Department of Neuroscience Rita Levi Montalcini and Neuroscience Institute Cavalieri Ottolenghi (NICO), Orbassano, Torino, Italy.
3: University of Milan, Department of Biosciences, Laboratory of Stem Cell Biology and Pharmacology of Neurodegenerative Diseases, Milan, Italy.
4: IRCCS Istituto delle Scienze Neurologiche di Bologna, Bellaria Hospital, Bologna, Italy.
5: Department of Pharmaceutics, Utrecht Institute for Pharmaceutical Sciences (UIPS), Utrecht University, Universiteitsweg 99, CG, Utrecht, The Netherlands.
6: Department of Molecular Medicine and Medical Biotechnology, University of Naples 'Federico II', Naples, Italy.
7: University of Bologna, Department of Biomedical and Neuromotor Sciences, Bologna, Italy.
8: University of Trento, Centre for Integrative Biology (CIBIO), Laboratory of Computational Oncology, Trento, Italy.
9: National Institute of Molecular Genetics (INGM) Romeo and Enrica Invernizzi, Milano, Italy.
10: Città della Salute e della Scienza University Hospital, Medical Genetics Unit, Torino, Italy.

Abstract

Allele-specific silencing by RNA interference (ASP-siRNA) holds promise as a therapeutic strategy for downregulating a single mutant allele with minimal suppression of the corresponding wild-type allele. This approach has been effectively used to target autosomal dominant mutations and single nucleotide polymorphisms linked with aberrantly expanded trinucleotide repeats. Here, we propose ASP-siRNA as a preferable choice to target duplicated disease genes, avoiding potentially harmful excessive downregulation. As a proof-of-concept, we studied autosomal dominant adult-onset demyelinating leukodystrophy (ADLD) due to lamin B1 (LMNB1) duplication, a hereditary, progressive and fatal disorder affecting myelin in the CNS. Using a reporter system, we screened the most efficient ASP-siRNAs preferentially targeting one of the alleles at rs1051644 (average minor allele frequency: 0.45) located in the 3' untranslated region of the gene. We identified four siRNAs with a high efficacy and allele-specificity, which were tested in ADLD patient-derived fibroblasts. Three of the small interfering RNAs were highly selective for the target allele and restored both LMNB1 mRNA and protein levels close to control levels. Furthermore, small interfering RNA treatment abrogates the ADLD-specific phenotypes in fibroblasts and in two disease-relevant cellular models: murine oligodendrocytes overexpressing human LMNB1, and neurons directly reprogrammed from patients' fibroblasts. In conclusion, we demonstrated that ASP-silencing by RNA interference is a suitable and promising therapeutic option for ADLD. Moreover, our results have a broad translational value extending to several pathological conditions linked to gene-gain in copy number variations.