qubit high sensitivity double stranded dna dsdna quantification kit  (Thermo Fisher)

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: The concentration of isolated genomic DNA was quantified by high-sensitivity Qubit dsDNA quantification assay kit from Thermo Fisher Scientific (Cat. No. Q33230 ).

Techniques: Biomarker Discovery, Luciferase, Transfection, Quantitative Proteomics, Two Tailed Test, Standard Deviation

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: Bxb1-mediated seamless insertion with AAV.AD in PHH and mice (A) Schematic of AAV.AD transfer plasmids designed for transduction reporter and integration studies. (B) Native agarose gel electrophoresis of packaged AAV.AD genomes compared to transfer plasmid controls with or without exonuclease treatment to determine DNA type. Table represents exonuclease activity on DNA substrate type, and UT represents untreated conditions. AAV.AD double-stranded pDNA is shown in both linearized and supercoiled (sc) states and expected to be 4.6 kb. Purified AAV.AD genomes are expected to be single-stranded with a length of 4,600 nts. DNA ladder represents dsDNA fragments. (C) AAV.AD transduction efficiency in HEK293 and PHH using promoter-driven NanoLuc and promoterless control reporters. (D) The effect of modulating AAV.AD transduction timing on I-PGI outcome at the PAH locus in PHH. Transduction timing represents hours prior to single-dose I-PGI transfection of synthetic atgRNAs, nCas9-RT, and Bxb1 mRNAs. (E) Expanded view of AAV.AD seamless integration alignment in PHH where each line represents an individual read with alignment (gray). (F) Schematic overview of study design to evaluate insertion efficiency of AAV.AD compared to scAAV in mice. (G) Quantification of AAV.AD and scAAV insertion at the mouse F9 locus ( n = 3 per group) where each data point reflects a single animal. Statistical significance was calculated using Student’s unpaired two-tailed t test (ns indicates not significant, ∗ p < 0.05). (H) Graphic illustration of seamless insertion outcome with AAV.AD. For (D and G), data were collected using ddPCR, and values were calculated as described in . Data reflect the mean and standard deviation of 2–3 biological replicates.

Article Snippet: The concentration of isolated genomic DNA was quantified by high-sensitivity Qubit dsDNA quantification assay kit from Thermo Fisher Scientific (Cat. No. Q33230 ).

Techniques: Transduction, Agarose Gel Electrophoresis, Plasmid Preparation, Activity Assay, Purification, Control, Transfection, Two Tailed Test, Standard Deviation