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: Journal of neurochemistry

Article Title: Neuronal filopodium formation induced by the membrane glycoprotein M6a (Gpm6a) is facilitated by coronin-1a, Rac1, and p21-activated kinase 1 (Pak1).

doi: 10.1111/jnc.13552

Figure Lengend Snippet: Fig. 1 (a) Gpm6a and Rac1 co-immunoprecipitate with Coro1a from rat hippocampal lysates. Western blot of proteins co-immunoprecipi- tated from rat hippocampal lysates using anti-Coro1a antibody and probed with anti-Gpm6a, anti-Rac1 and anti-Coro1a antibodies. For this purpose, the membrane (different kDa areas) was cut and incubated with indicated antibodies. Non-immune rabbit serum was used as a control. Gpm6a and Rac1 are present in the anti-Coro1a immunoprecipitate. Bands representing Gpm6a are indicated by stars. (b) Coro1a co-localizes with Gpm6a in hippocampal neurons. Confocal image of hippocampal neurons (4 DIV) co-immunostained with anti- bodies against Gpm6a (red), Coro1a (green) and dendritic marker MAP2 (blue). A portion of Gpm6a-labeled spots co-localizes with Coro1a (arrowheads; insets 1 and 2). Scale bar, 20 lm.

Article Snippet: Mammalian expression plasmids: pRFP-C1 encoding the red fluorescent protein (RFP), pEGFP-C1 (Clontech Laboratories, © 2016 International Society for Neurochemistry, J. Neurochem. (2016) 137, 46--61 Mountain View, CA, USA) encoding the enhanced green fluorescent protein (EGFP), RFP-tagged wild-type (wt) Gpm6a (Gpm6aRFP) described previously (Alfonso et al. 2005), EGFP-tagged wt Coro1a (wtCoro1a-EGFP), and a deletion mutant containing only 5 WD repeats (aa 65-306) of Coro1a [Coro1a(WD1-5)-EGFP] kindly provided by Dr William Trimble (Yan et al. 2005), EGFPtagged Rac1 T17N [Rac1DN-EGFP; Addgene#12982 (Subauste et al. 2000)], EGFP-tagged Rac1 Q61L [Rac1CA-EGFP; Addgene#12968 (Subauste et al. 2000)], myc-tagged Pak1 K299R [Addgene#12210 (Sells et al. 1997)], and myc-tagged Pak1 H83L/H86L [Addgene#12211 (Sells et al. 1997)]. siRNAs: siGENOME non-targeting siRNA Pool #2, Coro1a siRNAs targeting either its coding DNA sequence (CDS) [prevalidated in Suo et al. (Suo et al. 2014)] or its 30-UTR region (custom designed).

Techniques: Western Blot, Membrane, Incubation, Control, Marker, Labeling