Blood biodistribution and vector shedding of valoctocogene roxaparvovec in people with severe hemophilia A

• After valoctocogene roxaparvovec treatment, total and encapsidated vector DNA are steadily cleared from the blood and shedding matrices

• Risk of horizontal or germline transmission following valoctocogene roxaparvovec is extremely low

Following systemically-administered adeno-associated virus (AAV) gene therapy, vector particles are widely distributed, which has raised concerns about horizontal or germline transmission of vector. Characterization of biodistribution and kinetics of vector DNA in body fluids can address these concerns and provide insights into vector behavior in accessible samples. We investigated the biodistribution and vector shedding profile of valoctocogene roxaparvovec in men with severe hemophilia A enrolled in the phase 3 GENEr8-1 trial. Participants (n=134) received a single 6E13 vg/kg infusion and were assessed over 3 years. Vector DNA was measured with 4 different assays. Total vector DNA was evaluated in blood, saliva, stool, semen, and urine by quantitative (q)PCR. Encapsidated vector DNA was measured in plasma and semen with immunocapture-based qPCR. Contiguity of vector genomes and assembly of inverted terminal repeat fusions were measured in whole blood and peripheral blood mononuclear cells (PBMCs) using multi-color digital PCR. Median peak vector DNA levels observed 1 to 8 days after dosing were highest in blood, followed by saliva, semen, stool, and urine. Concentrations then declined steadily. Encapsidated vector DNA cleared faster than total vector DNA, achieving clearance by <=12 weeks in plasma and semen. Predominant vector genome forms transitioned from non-contiguous to full-length over time in whole blood and PBMCs, indicating formulation of stable circularized episomes within nucleated cells. The replication-incompetent nature of valoctocogene roxaparvovec, coupled with the steady clearance of total and encapsidated vector DNA from shedding matrices, indicates risk of transmission is low. This trial is registered at www.clinicaltrials.gov as NCT03370913.