Journal: Molecular Therapy. Nucleic Acids

Article Title: Rescue of the endogenous FVIII expression in hemophilia A mice using CRISPR-Cas9 mRNA LNPs

doi: 10.1016/j.omtn.2024.102383

Figure Lengend Snippet: Optimization of MC3 lipid nanoparticles (A) Gel electrophoresis analysis of MC3 lipid nanoparticles (LNPs) at different N/P ratios. Arrowheads and arrows indicate nicked form and supercoiled form of double-strand DNA, respectively. (B) Encapsulation efficiency of luciferase mRNA LNP (Luc LNP) at N/P ratios of 3 and 8, determined by RiboGreen assay. (C) Evaluation of luciferase expression 6 h after intravenous injection of 0.3 mg/kg Luc LNPs at N/P ratios of 3 and 8 using IVIS imaging. (D) Quantification of luciferase expression from IVIS imaging, presented as mean luciferase signals ± standard deviations, expressed in bioluminescence light units (BLUs), N/P 3, n = 3; N/P 8, n = 6. Characterization of LNP: (E) size distribution and (F) average size at N/P ratio = 8 was analyzed using qNano. Examination of plasma (G) AST and (H) ALT levels 1 day after intravenous injection of 1 mg/kg Luc LNP.

Article Snippet: D-Lin-MC3-DMA (MC3) was purchased from Chemscene (Monmouth Junction, NJ, USA) or MedKoo (Morrisville, NJ, USA).

Techniques: Nucleic Acid Electrophoresis, Encapsulation, Luciferase, Expressing, Injection, Imaging

Journal: Molecular Therapy. Nucleic Acids

Article Title: Rescue of the endogenous FVIII expression in hemophilia A mice using CRISPR-Cas9 mRNA LNPs

doi: 10.1016/j.omtn.2024.102383

Figure Lengend Snippet: Cell distribution of MC3 LNPs after intravenous injection in mice (A) One hour after intravenous injection of DiI-labeled LNPs (red) in mice, liver tissues were harvested and stained with FITC-Isolectin-B4 (green) to mark vascular cells. Colocalization of LNPs and vascular cells were indicated by white arrowheads. (B) To determine the LSEC-targeting ability, mice were intravenously injected with 1.2 mg/kg of GFP LNPs. Liver sections harvested 6 h after LNP injection were stained with anti-GFP (green) and anti-LYVE-1(red) antibodies. Co-stained cells were highlighted using white arrowheads. Enlarged images of the staining within white squares are presented below. (C) Mice were treated with 0.2 mg/kg Luc LNPs. Livers of treated mice were perfused with Liberase to obtain single cell suspensions. The total liver cells were subsequently purified to isolate hepatocytes (HPCs) and LSECs. Luciferase expression of HPCs and LSECs were determined by luciferase assay and normalized by total protein, respectively. Data are presented as average experimental values ± standard deviation.

Article Snippet: D-Lin-MC3-DMA (MC3) was purchased from Chemscene (Monmouth Junction, NJ, USA) or MedKoo (Morrisville, NJ, USA).

Techniques: Injection, Labeling, Staining, Purification, Luciferase, Expressing, Standard Deviation