Extended Data Fig. 3: Quantification of circular vector genomes [DNA] and assessment of multimeric circular episomal forms in liver samples from patients.
Quantification of circular vector genomes [DNA] as measured by ddPCR utilizing primers and probes to detect specific regions of the vector genome: a) R2-R10 linked amplicons. b) R1-R11 linked amplicons (full-length). c) SQ amplicon (representing overall vector genome count). d) ITR-fused amplicons; ITR fusion assay measured 5′ to 3′ ITR recombination (that is head-to-tail, H-T). e) Circular multimeric episome quantification following PS-DNase (eliminates all linear forms of DNA) or PS-DNase/KpnI digestion (to separate individual vector genome units within concatemeric circular forms). ITR fusion assay measured 5′ to 3′ ITR recombination (that is head-to-tail, H-T). f) Qualitative Southern blot assessment of vector genome configuration in circular forms obtained after DNA treatment with PS-DNase and KpnI. DNA fragments corresponding to 3.5 kb, or 4.4 and 2.6 kb could be detected for H-T or H-H/T-T concatemer configurations, respectively. Biopsies from Control, Participant 1, and Participant 11, and for Participants 3, 4, and 15 were processed at separate times; results are presented on separate blots from two independent experiments, and are not intended to present a quantitative comparison. ddPCR, droplet digital polymerase chain reaction; H, head (5′ end); H-H, head-to-head orientation; H-T, head-to-tail orientation; ITR, inverted terminal repeat; kb, kilobase pairs; LM, linear markers; PS-DNase, Plasmid Safe™ ATP-Dependent DNAase; T, tail (3′ end); vg, vector genome. ddPCR was performed after DNA digestion with PS-DNase and KpnI (to eliminate linear forms and to separate individual vector genome units within concatemeric circular forms).
