Adeno-associated virus (AAV)-based gene therapy vectors are replication-incompetent and thus pose minimal risk for horizontal transmission or release into the environment. In studies with AAV5-FVIII-SQ (valoctocogene roxaparvovec), an investigational gene therapy for hemophilia A, residual vector DNA was detectable in blood, secreta, and excreta, but it remained unclear how long structurally intact AAV5 vector capsids were present. Since a comprehensive assessment of vector shedding is required by regulatory agencies, we developed a new method (termed iqPCR) that utilizes capsid-directed immunocapture followed by qPCR amplification of encapsidated DNA. The limit of detection for AAV5 vector capsids was 1.17E+04 and 2.33E+04 vg/mL in plasma and semen, respectively. Acceptable precision, accuracy, selectivity, and specificity were verified; up to 1.00E+09 vg/mL non-encapsidated vector DNA showed no interference. Anti-AAV5 antibody plasma concentrations above 141 ng/mL decreased AAV5 capsid quantification, suggesting that iqPCR mainly detects free capsids and not those complexed with antibodies. In a clinical study, AAV5-FVIII-SQ capsids were found in plasma and semen but became undetectable within nine weeks after dose administration. Hence, iqPCR monitors the presence and shedding kinetics of intact vector capsids following AAV gene therapy and informs the potential risk for horizontal transmission.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) is an adeno-associated virus serotype five gene therapy under investigation for the treatment of hemophilia A. Herein, we assessed the potential for germline transmission of AAV5-hFVIII-SQ in mice. Male B6.129S6-Rag2tm1Fwa N12 mice received a single intravenous dose of vehicle or 6 × 1013 vg/kg AAV5-hFVIII-SQ. Vehicle and AAV5-hFVIII-SQ-treated mice were mated with naïve females 4 days after dosing, when the concentration of vector genomes was expected to be at its peak in semen, and 37 days after dosing, when a full spermatogenesis cycle was estimated to be complete. Quantitative PCR was used to evaluate the presence of transgene DNA in liver and testes from F0 males dosed with AAV5-hFVIII-SQ and liver tissue of F1 offspring. Transgene DNA was detected in liver and testes of all F0 males dosed with AAV5-hFVIII-SQ, confirming successful transduction. Importantly, no transgene DNA was detected in any tested F1 offspring derived from F0 males dosed with AAV5-hFVIII-SQ. Using a novel 2-stage statistical model that takes into account the number of males dosed with AAV5-hFVIII-SQ and the number of offspring sired by these males, we estimate that the risk of germline transmission is <5% with a 99.2% confidence level.

Introduction: Long-term durable expression of hFVIII-SQ has been observed following BMN 270 (AAV5-hFVIII-SQ, valoctocogene roxaparvovec) single-dose administration in patients with severe hemophilia A. Although adeno-associated virus (AAV) vectors are replication incompetent and thus pose minimal risk for transmission or release into environment, a comprehensive assessment of vector shedding in secreta and excreta is required as part of the clinical development program. In addition, evaluation of vector biodistribution in blood is useful to characterize vector DNA processing and further understand the kinetics of vector DNA clearance. Vector shedding and biodistribution were evaluated from subjects from an ongoing Phase 1/2 study (Study 270-201, NCT02576795) and an ongoing Phase 3 study (Study 270-301, NCT03370913) following BMN 270 administration in patients with severe hemophilia A. Methods: In the Phase 1/2 study, 15 adult male subjects with severe hemophilia A received a single intravenous infusion of 6E12 vg/kg (n=1), 2E13 vg/kg (n=1), 4E13 vg/kg (n=6), or 6E13 vg/kg (n=7) BMN 270. In the Phase 3 study, 134 adult male subjects with severe hemophilia A received a single intravenous infusion of 6E13 vg/kg BMN 270. In both studies, measurement of vector DNA in blood, saliva, feces, semen, and urine was performed using a validated qPCR assay. Blood, saliva, urine, stool, and semen were collected until at least 3 consecutive negative results via qPCR were obtained. To further characterize vector DNA potentially capable of cell transduction, a novel immunocapture qPCR (iqPCR) assay was developed to measure the amount of intact AAV5 vector capsids in plasma and semen. Further assessments of the biodistribution of vector DNA in blood, including the evaluation of the contiguity and structural characteristics of BMN 270 vector genomes, were performed in blood, plasma, peripheral blood mononuclear cells (PBMC), and red blood cells using a drop-phase droplet-digital (dd)PCR assay. Results: Following BMN 270 administration at all dose levels, vector DNA was detected in all subjects in all biodistribution and shedding matrices evaluated (i.e., blood, saliva, urine, stool, and semen). Median peak vector DNA levels were greatest in blood followed by saliva, semen, stool, and urine. Peak vector DNA concentrations following BMN 270 administration were observed early. Following peak vector DNA concentrations, BMN 270 vector genomes were steadily cleared from the urine, semen, saliva, stool, and blood. In comparison to total vector DNA measured by qPCR, encapsidated vector DNA in plasma and semen was cleared more rapidly, as measured using iqPCR. Evaluation of total vector DNA in whole blood and blood fractions, indicate 3 phases of vector DNA clearance, which are associated with the expected lifespan of various transduced cell types. From approximately 24 weeks after BMN 270 administration and beyond, a slower rate of decline of vector DNA in whole blood is observed with the majority of transgene DNA present beyond 24 weeks in blood likely within the PBMC fraction. Further characterization of vector DNA in blood demonstrated that BMN 270 DNA transitioned from an initial truncated form into full-length transgenes over time. In addition, the fraction of DNA detected in whole blood that contains an inverted terminal repeat (ITR) fusion, indicating that the residual vector DNA may have formed circular episomes in the transduced cells, increased over time. By 52 weeks post-BMN 270 administration, the majority of vector DNA in whole blood was full-length and contained an ITR fusion. Conclusions: Vector shedding and distribution has been extensively evaluated in patients with severe hemophilia A treated with BMN 270. Both vector DNA and vector capsids were detected and steadily cleared in blood and shedding matrices. Based upon the replication incompetent nature of BMN 270 and the maximum potential exposure to the vector in secreta and excreta following BMN 270 administration, the risk of transmission to untreated individuals is considered extremely low. The biodistribution and characterization of vector DNA in blood cells demonstrates the formation of full-length transgenes with ITR fusions. Disclosures Clark: BioMarin Pharmaceutical In.: Current Employment. Hammon:BioMarin Pharmaceutical Inc.: Current Employment. Sandza:BioMarin Pharmaceutical Inc.: Current Employment. Torres:BioMarin Pharmaceutical Inc.: Current Employment. Koziol:BioMarin Pharmaceutical Inc.: Current Employment. Holcomb:BioMarin Pharmaceutical Inc.: Current Employment. Kim:BioMarin Pharmaceutical Inc.: Current Employment. Jayaram:BioMarin Pharmaceutical Inc.: Current Employment. Russell:BioMarin Pharmaceutical Inc.: Current Employment, Current equity holder in publicly-traded company; Amgen nc.: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Vettermann:BioMarin Pharmaceutical Inc.: Current Employment. Henshaw:BioMarin Pharmaceutical Inc.: Current Employment.