Journal: Nucleic Acids Research

Article Title: Development of a DUX4 -targeting antibody oligonucleotide conjugate as a therapy for FSHD

doi: 10.1093/nar/gkag301

Figure Lengend Snippet: Illustration of AOC 1020 structure and activity evaluation of DUX4-targeted siRNAs in myotubes derived from FSHD patients. ( A ) Scheme of the AOC 1020 structure (drug-antibody ratio equal to 1). siDUX4.6 modification pattern is outlined in . ( B ) Primary FSHD patient-derived myoblasts were transfected with DUX4 -targeting siRNAs and induced to differentiate into myotubes to perform gene expression analysis. The FSHD composite score integrates the gene expression levels of four DUX4-regulated genes ( MBD3L2, ZSCAN4, LEUTX , and KHDC1L ). Screening of the DUX4 siRNA library was performed in FSHD1 patient-derived primary myotubes MB06 at 10 nM final concentration. Data are represented as a percentage of mock transfection control (mean ± SD, n = 4 for library siRNAs, n = 8 for controls). ( C ) Activity of siDUX4.6 was evaluated across nine FSHD donor myotubes (MB06 data are the same as in panel B) (mean ± SD; n = 4 for siRNAs; n = 8 for mocks for MB03, MB04, and MB10; n = 4 for mocks for MB01, MB05, MB11, and MB12). Myotubes were collected 2 days (MB01, MB03), 3 days (MB02, MB05, MB07, MB10, MB11, MB12), or 4 days (MB04, MB06) after inducing differentiation ( D ). In vitro potency of the siDUX4.6 sequence was evaluated by concentration-response in two FSHD1 patient-derived primary myotubes (MB05, MB06). Data in the graphs are represented as a percentage of mock transfection control (mean ± SD, n = 4 for siRNA, n = 28 for mock). Log(inhibitor) versus response three-parameter calculation was used to fit the concentration-response curves. The best-fitting values for EC 50 (half maximal effective concentration) and E max (maximum response) are reported in the table. CTRL, control; FSHD, facioscapulohumeral muscular dystrophy; NTC, non-targeting control; siRNA, small interfering ribonucleic acid.

Article Snippet: Myotubes were collected in TRIzol and stored at −80°C until processing for RNA isolation using Direct-zol-96 RNA isolation kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol.

Techniques: Activity Assay, Derivative Assay, Modification, Transfection, Gene Expression, Concentration Assay, Control, In Vitro, Sequencing

Journal: Nucleic Acids Research

Article Title: Development of a DUX4 -targeting antibody oligonucleotide conjugate as a therapy for FSHD

doi: 10.1093/nar/gkag301

Figure Lengend Snippet: Transcriptome-wide effect of siDUX4.6 on DUX4-regulated genes in FSHD myotubes. Differential gene expression data are presented as volcano plots. One healthy (MB07) and three FSHD patient-derived primary myoblast cell lines (MB02, MB05, MB06) were treated with 10 nM siDUX4.6. Myotubes were collected in Trizol 3 days (MB02, MB05, MB07) or 4 days (MB06) after inducing differentiation, and gene expression was analyzed by RNA sequencing. The significance was established as FDR<0.05. ( A ) Average differential gene expression of three FSHD patient myotubes compared with one healthy donor myotube is plotted, all samples treated with non-targeting control siRNA. ( B ) Average differential gene expression of three FSHD patient myotubes treated with 10 nM siDUX4.6 versus non-targeting control siRNA is plotted. n = 4 for treatment in each individual cell line. Arrows at the bottom of the graphs indicate if genes were upregulated or downregulated. DE, differentially expressed; FC, fold-change; FDR, false discovery rate; FSHD, facioscapulohumeral muscular dystrophy; siRNA, small interfering ribonucleic acid.

Article Snippet: Myotubes were collected in TRIzol and stored at −80°C until processing for RNA isolation using Direct-zol-96 RNA isolation kit (Zymo Research, Irvine, CA, USA) following the manufacturer’s protocol.

Techniques: Gene Expression, Derivative Assay, RNA Sequencing, Control