Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Therapeutic mRNA delivery of CRISPR-Cas9 to the trabecular meshwork reverses ocular hypertension in myocilin glaucoma

doi: 10.1016/j.omtm.2025.101614

Figure Lengend Snippet: Lipoplex encapsulating Cas9-mRNA and gRNA targeting MYOC reduces intracellular accumulation of mutant myocilin protein in cultured TM cells GTM3 cells stably expressing mutant MYOC were transfected with lipoplexes containing either Cas9 mRNA + scrambled gRNA (Control) or Cas9 mRNA + MYOC -targeting gRNA (Treated). Immunostaining for (A) KDEL or (B) Protein disulfide isomerase (PDI) revealed a reduction in intracellular accumulation of mutant myocilin protein in the ER and its associated ER stress in treated cells compared to controls. N = 3. (C) Western blot analysis of MYOC and GRP78 in cell lysates from TM cells expressing mutant MYOC treated with either Cas9 mRNA + scrambled gRNA (Control) or Cas9 mRNA + MYOC -targeting gRNA (Treated). (D) Densitometric analysis of the Western blots revealed a significant reduction in MYOC protein levels in CRISPR-Cas9-treated cells compared to controls. GRP78, an ER stress marker, was reduced significantly upon Cas9 mRNA + MYOC -targeting gRNA treatment compared to controls, ( n = 7 MYOC, n = 4 KDEL, ∗∗∗∗ p < 0.0001).

Article Snippet: Fixed cells were blocked with 10% goat serum in 0.1% Triton X-100 for 2 h. Primary antibodies against MYOC (Catalog #60357, Proteintech) and KDEL (Catalog #NBP1-97469, Novus) were incubated overnight, followed by four washes with 1x PBS.

Techniques: Mutagenesis, Cell Culture, Stable Transfection, Expressing, Transfection, Control, Immunostaining, Western Blot, CRISPR, Marker

Journal: Molecular Therapy. Methods & Clinical Development

Article Title: Therapeutic mRNA delivery of CRISPR-Cas9 to the trabecular meshwork reverses ocular hypertension in myocilin glaucoma

doi: 10.1016/j.omtm.2025.101614

Figure Lengend Snippet: Cas9 mRNA and gRNA targeting MYOC rescues the glaucomatous pathology induced by mutant MYOC in the TM of Tg.CreMYOC Y437H mice (A) Tg.CreMYOC Y437H mice were injected with lipoplex-Cre mRNA along with Cas9 mRNA+scrambled gRNA or gRNA targeting MYOC . IOP was measured weekly. Tg.CreMYOC Y437H mice injected with Cre and Cas9+scrambled gRNA developed a significant IOP elevation 3 weeks post-injection and sustained IOP elevation throughout the study. Tg.CreMYOC Y437H mice injected with lipoplex loaded with Cre and Cas9 mRNA+gRNA targeting MYOC did not show changes in IOPs, indicating rescue of ocular hypertension in Tg.CreMYOC Y437H mice. Two-way ANOVA with repeated measurements and Bonferroni post-hoc analysis were performed. Data represented as mean ± SEM; ∗∗ p < 0.01, and ∗∗∗ p < 0.001. Representative immunostaining for MYOC and KDEL (B) and its quantification (C) demonstrated that Cas9 mRNA+g MYOC significantly reduces MYOC and its co-localization with the ER marker KDEL in the TM of Tg.CreMYOC Y437H mice compared to control-treated mice. Quantification revealed a 54% reduction in MYOC levels and a 55% reduction in KDEL levels following Cas9/gRNA treatment ( n = 4, ∗∗∗∗ p < 0.0001).

Article Snippet: Fixed cells were blocked with 10% goat serum in 0.1% Triton X-100 for 2 h. Primary antibodies against MYOC (Catalog #60357, Proteintech) and KDEL (Catalog #NBP1-97469, Novus) were incubated overnight, followed by four washes with 1x PBS.

Techniques: Mutagenesis, Injection, Immunostaining, Marker, Control

Journal: bioRxiv

Article Title: ncBAF, a chromatin remodeler, enhances PXR-mediated transcriptional activation in the human and mouse liver

doi: 10.1101/2023.02.03.527063

Figure Lengend Snippet: Effects of iBRD9 on the induction of CYP3A in mouse liver. C57BL/6J or hCYP3A-MAC/hPXR male mice (n = 6) were intraperitoneally treated with 50 mg/kg PCN or 10 mg/kg rifampicin for four consecutive days and intraperitoneally treated with 10 mg/kg iBRD9 every other day. Cyp3a11, Cyp3a25, CYP3A4, and Gapdh mRNA (A, D), and Cyp3a, CYP3A4, and KDEL protein (B, E) levels were evaluated by real-time RT□PCR and Western blotting, respectively. (C, F) Triazolam α- and 4-hydroxylase activities were evaluated as marker activity for Cyp3a and CYP3A4. Each column represents the mean ± SD (n = 6). n refers to biological repeats. ** P < 0.01 and *** P < 0.001, compared with vehicle, † P < 0.05 and †† P < 0.01, compared with iBRD9 (−). The experiments were repeated two times with similar results.

Article Snippet: Rabbit anti-human GAPDH polyclonal antibody (NB100-56875) and mouse anti-KDEL monoclonal antibody (NBP1-97469) were purchased from Novus Biologicals (Centennial, CO).

Techniques: Western Blot, Marker, Activity Assay