Background: Valoctocogene roxaparvovec is a rAAV5-FVIII-SQ gene therapy investigated for treatment of hemophilia A. While the target organ for expression of FVIII is liver, small amounts of vector DNA in blood provide insight into kinetics of vector DNA structures in recipients’ cells.

Aims: Evaluate valoctocogene roxaparvovec genome kinetics in blood.

Methods: DNA was extracted from whole blood or fractionated blood, from participants in valoctocogene roxaparvovec phase 1/2 and phase 3 studies. Analyses with multicolor ddPCR, qPCR, and ITR-fusion ddPCR assays were used to investigate vector DNA presence, contiguity, and circularization.

Results: Total vector DNA in blood decreased five orders of magnitude over the first 26 weeks post-administration, with distinct phases corresponding to lifespans of granulocytes and red blood cells, while very small amounts remained in the peripheral blood mononuclear cell (PBMC) fraction. Post-administration, only small amounts of vector DNA could be found unassociated with cells in plasma, and was largely fragmented. At six months, remaining vector DNA in the PBMC fraction (the only long-lived, nucleated cells) had largely undergone processing into assembled full-length transgenes, and ITR fusion consistent with stable episomal structures, while DNA in other cell fractions was either unprocessed or fragmented. Low level vector DNA was detectable past two years in the phase 1/2 study, consistent with long-lived PBMC lifespans. Moreover, DNA structural kinetics were consistent between recipients across phase 1/2 and phase 3 studies, within the first year of available samples.

Conclusions: Blood offers an accessible and relatively non-invasive window into rAAV vector molecular processing in human cells. Vector DNA in blood, outside of PBMCs, became largely degraded confirming negligible risk of horizontal transmission from blood. In the blood, nucleated PBMCs processed valoctocogene roxaparvovec vector into full-length transgenes, and maintained stable, durable forms for long periods of time. Reproducible results across clinical phase studies help support comparability of product throughout clinical development.

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ISTH Congress Abstracts - https://abstracts.isth.org/abstract/consistent-structural-kinetics-of-valoctocogene-roxaparvovec-dna-in-human-blood-samples/