Journal: Molecular Therapy. Methods & Clinical Development
Article Title: Development of circular AAV cargos for targeted seamless insertion with large serine integrases
doi: 10.1016/j.omtm.2025.101490
Figure Lengend Snippet: Validation of cAAV in primary human hepatocytes (A) Schematic of cAAV design and mechanism. Orthogonal attachment sites (attB∗, attP∗) are added to an attP AAV genome to facilitate Bxb1-mediated intramolecular recombination. The reaction produces a circular dsDNA attP cargo and an ITR by-product. (B) Design of a ssAAV dual luciferase circularization reporter to validate cAAV function. In linear native format, EF1A promoter expresses Firefly luciferase, and NanoLuc is not expressed. When circularized, EF1A promoter expresses NanoLuc in the circular episome and Firefly luciferase is not expressed in the ITR by-product. (C) Relative amounts of linear or circularized cAAV reporter in PHH on days 1 and 5 following Bxb1 mRNA transfection. (D) Absolute quantification of cAAV circularization frequency on day 5 following Bxb1 mRNA transfection in PHH. (E) I-PGI outcome with cAAV or scAAV cargo over a 6-day time course in PHH. Statistical significance was calculated using Student’s unpaired two tailed t test (ns indicates not significant, ∗ p < 0.05, ∗∗ p < 0.01). (F) The effect of staggering cargo circularization and I-PGI on seamless insertion junction outcome in PHH. Predose is Bxb1 mRNA transfected twice, on day 2 and on day 5. For E-F, I-PGI was performed at the PAH locus through a single transfection of synthetic atgRNA, nCas9-RT and Bxb1 mRNAs. For (D–F), data were collected using ddPCR, and values were calculated as described in . Data reflect the mean and standard deviation of three biological replicates.
Article Snippet: Cryopreserved primary human hepatocytes (Thermo Fisher) were recovered in Cryopreserved Hepatocyte Recovery Media (Gibco CM7000) and plated at 37–42k cells per well.
Techniques: Biomarker Discovery, Luciferase, Transfection, Quantitative Proteomics, Two Tailed Test, Standard Deviation
