Journal: bioRxiv

Article Title: Enabling non-viral DNA delivery using lipid nanoparticles co-loaded with endogenous anti-inflammatory lipids

doi: 10.1101/2024.06.11.598533

Figure Lengend Snippet: a, b , Design of experiment (DoE) screening to optimize NOA-pDNA-LNP formulation in RAW264.7 cells. Using JMP software, a full factorial screen was designed by varying ionizable lipid mol% (30 to 50), total lipid to pDNA w/w ratio (10:1 to 40:1), and the type of helper lipid used (DSPC, DOPE, DOTAP, and 18:0 PG). All LNPs contained 0.2 D/L of NOA. 24-hour after 500 ng/mL treatment in RAW264.7 cells, luciferase protein expression was measured highlighting the influence of LNP formation parameters for transgene expression ( a ). NOA-pDNA-LNP optimized with DOTAP as the helper lipid led to 50-fold increase in transgene expression, when compared to standard NOA-pDNA-LNP ( b ). c, d . 2D monoculture of difficult-to-transfect cell line, human induced-pluripotent-stem-cell-derived type II alveolar epithelial cells (iAT2s), were treated with 1000 ng of eGFP encoding pDNA - using lipofectamine, standard NOA-pDNA-LNPs, or optimized NOA-pDNA-LNPs - and imaged after 48-hours. tdTomato (red) signal indicates iAT2 positively, while eGFP (green) signal indicates successfully transfected cells ( c ). 120-hours post transfection, tdTomato + cells that were also eGFP + were quantified using flow cytometry indicating similar transfection levels (trending higher) for optimized NOA-pDNA-LNPs to the gold standard, lipofectamine 2000 ( d ). Statistics : n=2 per LNP formulation for a (biological replicates), n=3/group for b and d (biological replicates). Data shown represents mean ± SEM.

Article Snippet: Lipofectamine 2000 (Thermo) transfection of eGFP pDNA was performed according to the manufacturer’s protocol. iAT2s were imaged for tdTomato retention and eGFP expression using an Eclipse Ti2 Series inverted microscope (Nikon) and 24-, 48-, and 120-hours after treatment.

Techniques: Formulation, Software, Luciferase, Expressing, Derivative Assay, Transfection, Flow Cytometry

Journal: bioRxiv

Article Title: miR-486 is an epigenetic modulator of Duchenne muscular dystrophy pathologies

doi: 10.1101/2021.06.14.448387

Figure Lengend Snippet: A. Schematic demonstrating the workflow for the chimeric eCLIP-sequencing platform to identify miR-486 in vivo skeletal muscle target transcripts. TA muscle was harvested from six-month-old WT and miR-486 KO male mice and total RNA isolation was completed. The Ago2-miR-486 complex bound to target RNA transcripts was isolated and then sequencing was performed to map the reads of transcripts bound to the Ago2-miR-486 complex. B. Chromosomal location of a single top “hit”, Mt2, as identified by eCLIP-seq as a direct target of miR-486. Peaks generated using Integrative Genome Viewer. C. Metagene plot demonstrating overall miR-486 binding location by relative position on target gene. D. Pie chart demonstrating the proportion of miR-486 gene targets and the respective intragenic binding location of miR-486. E. Table outlining the top 10 transcripts that were identified as direct targets of miR-486 via CLIP-seq. F. g:Profiler enrichment analysis graph demonstrates the most significant cellular pathways associated with the 18 direct miR-486 targets identified via chimeric eCLIP-seq. The pathway ID number in the table correlates with the numbered dots in the accompanying graph above. G. Quantitative PCR of 18 miR-486 eCLIP-seq targets in miR-486 KO and mdx 5cv TA muscles expression levels compared to WT controls in separate cohort analyses. Data points are individual biological replicates, n=4/cohort and logarithmic fold change normalized to both wild type and β-actin is shown. **p=≤0.01. mRNA levels are normalized to β-actin and miR-486 KO levels are shown as relative to WT.

Article Snippet: For the chimeric CLIP-sequencing we used adult 6-month-old male TA muscles from WT and miR-486 KO mice (n = 3 muscles for each cohort) that were snap frozen in liquid nitrogen and chimeric eCLIP-sequencing was performed with assistance by Eclipse BioInnovations (Eclipse BioInnovations; San Diego, CA).

Techniques: Sequencing, In Vivo, Isolation, Generated, Binding Assay, Real-time Polymerase Chain Reaction, Expressing