Journal: bioRxiv

Article Title: Potent and durable gene modulation in heart and muscle with chemically defined siRNAs

doi: 10.1101/2024.10.01.616183

Figure Lengend Snippet: a panel of 80-96 cholesterol conjugated siRNAs targeting (A) murine Mstn gene and (B) human MSTN gene were tested for their silencing ability using passive delivery in differentiated murine myoblasts for murine-targeting sequences, and Rhabdomyosarcoma cells for human-targeting sequences. Each screen was followed by measuring the dose-dependent silencing of three selected functional sequences for each species, to obtain the most potent sequence with the lowest IC50. (mRNA measured by the Quantigene 2.0 Assay, n=3, Data represented as mean± S.D.; dotted line on all graphs represents 25% remaining mRNA expression).

Article Snippet: mRNA expression was quantified with the QuantiGene Singleplex Assay Kit (Invitrogen # QS0016), a hybridization-based assay, as previously described ( , ).

Techniques: Functional Assay, Sequencing, Expressing

Journal: bioRxiv

Article Title: Potent and durable gene modulation in heart and muscle with chemically defined siRNAs

doi: 10.1101/2024.10.01.616183

Figure Lengend Snippet: (A) Schematic Mstn 1928 -DCA-siRNA chemical modifications used to stabilize the siRNA, with and without exNA terminal modification. (B) Mstn mRNA silencing in multiple tissues 1-week post injection, using the previously tested Mstn 1192 sequence with and without exNA inclusion, as well as comparing to the newly identified siRNA lead Mstn 1928 . (C) Mstn mRNA and MSTN protein silencing 2-weeks post injection at various escalating doses via single and dual injection. All study groups are n=4-5 mice/group. Statistical analysis was performed in GraphPad Prism: for section (B) one-way ANOVA analysis followed by Tukey’s multiple comparisons across all groups, with only significant comparisons against NTC-DCA-siRNA shown, as well as significant comparisons between the treated groups. For section (C) only comparisons to NTC-DCA-siRNA are shown. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). Subset of data of Mstn1192 from panel B was previously published in Yamada et. al . . (mRNA measured by the Quantigene 2.0 Assay; protein measured by ELISA; Data represented as mean± S.D.; dotted line on all graphs represents 25% remaining mRNA or protein expression).

Article Snippet: mRNA expression was quantified with the QuantiGene Singleplex Assay Kit (Invitrogen # QS0016), a hybridization-based assay, as previously described ( , ).

Techniques: Modification, Injection, Sequencing, Enzyme-linked Immunosorbent Assay, Expressing

Journal: bioRxiv

Article Title: Potent and durable gene modulation in heart and muscle with chemically defined siRNAs

doi: 10.1101/2024.10.01.616183

Figure Lengend Snippet: (A) Mstn 1928 -DCA-siRNA with various modification patterns and PS backbone at the 3’ end of guide strand were administered subcutaneously, with plasma being sampled weekly for 4 weeks. (B) MSTN protein levels in plasma over time show that the alternating pattern 2 and methyl rich pattern 3 outperform pattern 1 in maximum silencing and durability of consistent silencing over 4 weeks. (C) siRNA accumulation in Quadriceps, Gastrocnemius and Heart at 4 weeks post injection show no significant differences in tissue delivery. (D) Mstn mRNA silencing at 4 weeks is consistent with the MSTN protein levels observed, where pattern 2 with 4PS at the terminus of the guide strand shows the strongest silencing in muscle and heart. All study groups are n=4-5 mice/group. Statistical analysis was performed in GraphPad Prism: for MSTN plasma levels, two-way ANOVA analysis using mixed model was performed, and only significant comparisons are shown; for tissue accumulation and mRNA levels, one-way ANOVA analysis followed by Tukey’s multiple comparisons across all groups, with only significant comparisons against NTC DCA-siRNA shown, as well as significant comparisons between the 4PS groups. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). (mRNA measured by the Quantigene 2.0 Assay; protein measured by ELISA; Data represented as mean± S.D.; dotted line on all graphs represents 25% remaining mRNA or protein expression).

Article Snippet: mRNA expression was quantified with the QuantiGene Singleplex Assay Kit (Invitrogen # QS0016), a hybridization-based assay, as previously described ( , ).

Techniques: Modification, Injection, Enzyme-linked Immunosorbent Assay, Expressing

Journal: bioRxiv

Article Title: Potent and durable gene modulation in heart and muscle with chemically defined siRNAs

doi: 10.1101/2024.10.01.616183

Figure Lengend Snippet: (A) Mstn silencing is not limited to lower limb muscle tissues but also observed in upper limb, internal and oral muscles. Tissues were harvested from the 40 mg/kg/2wks cohort 3 weeks post last administration. (B) Potent silencing n muscle and heart is not limited to Mstn, as we observe more 75-90% silencing in quadriceps and 50-70% silencing in heart for 3 other targets: Jak1, Mecp2, and Htt. Different targets are separated by dotted vertical lines. All study groups are n=4-5 mice/group. Statistical analysis was performed in GraphPad Prism: one-way ANOVA analysis followed by Tukey’s multiple comparisons across all groups, with only significant comparisons against NTC-DCA-siRNA shown. (* p<0.05, ** p<0.01, *** p<0.001, **** p<0.0001). (mRNA measured by the Quantigene 2.0 Assay; Data represented as mean± S.D.; dotted line on all graphs represents 25% remaining mRNA or protein expression).

Article Snippet: mRNA expression was quantified with the QuantiGene Singleplex Assay Kit (Invitrogen # QS0016), a hybridization-based assay, as previously described ( , ).

Techniques: Muscles, Expressing