Journal: bioRxiv

Article Title: Efficient and durable gene activation by Cas9-mediated epigenome editing in vivo

doi: 10.1101/2024.05.03.592438

Figure Lengend Snippet: (A) Summary of gRNA screening results using cynomolgus primary skeletal myoblast cells. Relative LAMA1 mRNA level is determined by qPCR where each sample expression values were normalized to the NTG sample values. Bars represent mean values and SEM is represented by error bars (n = 3). (B) MYOAAV-GNDM-c58 (1 x 10 14 vg/kg) was administered by i.v. infusion to 2-year old cynomolgus macaques after standard immunosuppression(IS) treatment. Blood was sampled at indicated timepoint, and all animals were sacrificed 6 weeks after administration. Animal #2003 had AAV seroconversion before administration, thus excluded from analysis. (C) Quantification of serum CK, ALT and AST enzyme levels before and after systemic administration of MYOAAV-GNDM-c58. Data are presented as individual and mean value. (D) Anti-Cas9 and anti-MYOAAV antibody responses were analyzed by serum ELISA assay using plates coated with specific antigens and developed plates were scanned at 450nm. Data are presented as individual and mean OD at 450nm. (E) Cas9 specific T cell responses (IFNg ELISPOT assay) were analyzed at terminal time point. 125k PBMCs were re-stimulated with Cas9 protein (10ug/ml) or PHA (5ug/ml) for 48hrs and ELISPOT plate was developed. Data was presented as spot forming units per million PBMCs. No-stimulation condition was used to subtract the background. (F) GC tissues were sectioned for both H&E staining and immunofluorescent staining with anti-CD8 and anti-CD11b antibodies. Images were captured with ECHO Revolution color camera at 20x magnification. Scale bars, 100 µm scale. (G) RT-qPCR analysis of immune cell markers using cDNA derived from GC muscle. Gene expression was normalized by HPRT and presented as fold change relative to untreated animal samples. All values are obtained by averaging results from triplicates for each sample. Data are presented as individual and mean.

Article Snippet: Primary cynomolgus macaque skeletal muscle myoblast cells were obtained from BioIVT.

Techniques: Expressing, Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot, Staining, Quantitative RT-PCR, Derivative Assay

Journal: bioRxiv

Article Title: Efficient and durable gene activation by Cas9-mediated epigenome editing in vivo

doi: 10.1101/2024.05.03.592438

Figure Lengend Snippet: (A) MYOAAV-GNDM-c58 (5 x 10 13 vg/kg) was administered by i.v. infusion to 8-month old cynomolgus macaques after standard immunosuppression(IS) treatment. Blood was sampled at indicated timepoint, and all animals were sacrificed 13 weeks after administration. (B) Quantification of serum CK, ALT and AST enzyme levels before and after systemic administration of MYOAAV-GNDM-c58. Data are presented as individual and mean value. (C) Anti-Cas9 and anti-MYOAAV antibody responses were analyzed by serum ELISA assay using plates coated with specific antigens and developed plates were scanned at 450nm. Data are presented as individual and mean OD at 450nm.(D) Cas9 specific T cell responses (IFNg ELISPOT assay) were analyzed at terminal time point. 125k PBMCs were re-stimulated with Cas9 protein (10ug/ml) or PHA (5ug/ml) for 48hrs and ELISPOT plate was developed. Data was presented as spot forming units per million PBMCs. No-stimulation condition was used to subtract the background.

Article Snippet: Primary cynomolgus macaque skeletal muscle myoblast cells were obtained from BioIVT.

Techniques: Enzyme-linked Immunosorbent Assay, Enzyme-linked Immunospot