Cerliponase alfa is recombinant human tripeptidyl peptidase 1 (TPP1) delivered by i.c.v. infusion for CLN2, a pediatric neurodegenerative disease caused by deficiency in lysosomal enzyme TPP1. We report the pharmacokinetics (PK) and pharmacodynamics of cerliponase alfa, the first i.c.v. enzyme replacement therapy, characterized in a phase I/II study. Escalating doses (30-300 mg Q2W) followed by 300 mg Q2W for ≥ 48 weeks were administered in 24 patients aged ≥ 3 years. Concentrations peaked in cerebrospinal fluid (CSF) at the end of ~ 4-hour i.c.v. infusion and 8 hours thereafter in plasma. Plasma exposure was 300-1,000-fold lower than in CSF, with no correlation in the magnitude of peak concentration (C max) or area under the concentration-time curve (AUC) among body sites. There was no apparent accumulation in CSF or plasma exposure with Q2W dosing. Interpatient and intrapatient variability of AUC, respectively, were 31-49% and 24% in CSF vs. 59-103% and 80% in plasma. PK variability was not explained by baseline demographics, as sex, age, weight, and CLN2 disease severity score did not appear to impact CSF or plasma PK. No apparent correlation was noted between CSF or plasma PK and incidence of adverse events (pyrexia, hypersensitivity, seizure, and epilepsy) or presence of antidrug antibodies in CSF and serum. There was no relationship between magnitude of CSF exposure and efficacy (change in CLN2 score from baseline), indicating maximum benefit was obtained across the range of exposures with 300 mg Q2W. Data from this small trial of ultra-rare disease were leveraged to adequately profile cerliponase alfa and support 300 mg i.c.v. Q2W for CLN2 treatment. CLN2 disease is an ultra-rare, inherited, pediatric neurodegenerative disorder that belongs to the family of neuronal ceroid lipofuscinoses (NCL; number indicates the form of NCL), collectively and more familiarly known as Batten disease. 1,2,3 Classified by the underlying gene defect, CLN2 specifically, is caused by mutations in the gene encoding the lysosomal enzyme, tripeptidyl peptidase 1 (TPP1). TPP1 deficiency results in lysosomal accumulation of abnormal storage material (called ceroid and lipofuscin) in neuronal cells, leading to cell damage and cell death of mainly the central nervous system (CNS) and the retina. This rapidly progressing, irreversible disease typically appears

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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.

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.