Lack of germline transmission in male mice following a single intravenous administration of AAV5-hFVIII-SQ gene therapy.

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.

Young mice administered adult doses of AAV5-hFVIII-SQ achieve therapeutic factor VIII expression into adulthood.

Valoctocogene roxaparvovec (AAV5-hFVIII-SQ) gene transfer provided reduced bleeding for adult clinical trial participants with severe hemophilia A. However, pediatric outcomes are unknown. Using a mouse model of hemophilia A, we investigated the effect of vector dose and age at treatment on transgene production and persistence. We dosed AAV5-hFVIII-SQ to neonatal and adult mice based on body weight or at a fixed dose and assessed human factor VIII-SQ variant (hFVIII-SQ) expression through 16 weeks. AAV5-hFVIII-SQ dosed per body weight in neonatal mice did not result in meaningful plasma hFVIII-SQ protein levels in adulthood. When treated with the same total vector genomes per mouse as adult mice, neonates maintained hFVIII-SQ expression into adulthood, although plasma levels were 3- to 4-fold lower versus mice dosed as adults. Mice <1 week old initially exhibited high hFVIII-SQ plasma levels and maintained meaningful levels into adulthood, despite a partial decline potentially due to age-related body mass and blood volume increases. Spatial transduction patterns differed between mice dosed as neonates versus adults. No features of hepatotoxicity or endoplasmic reticulum stress were observed with dosing at any age. These data suggest that young mice require the same total vector genomes as adult mice to sustain hFVIII-SQ plasma levels.

Immunogenicity to cerliponase alfa intracerebroventricular enzyme replacement therapy for CLN2 disease: Results from a Phase 1/2 study.

Treatment with intracerebroventricular (ICV)-delivered cerliponase alfa enzyme replacement therapy (ERT) in a Phase 1/2 study of 24 subjects with CLN2 disease resulted in a meaningful preservation of motor and language (ML) function and was well tolerated. Treatment was associated with anti-drug antibody (ADA) production in the cerebrospinal fluid (CSF) of 6/24 (25%) and in the serum of 19/24 (79%) of clinical trial subjects, respectively, over a mean exposure of 96.4 weeks (range 0.1-129 weeks). Neutralizing antibodies (NAb) were not detected in the CSF of any of the subjects. No events of anaphylaxis were reported. Neither the presence of serum ADA nor drug-specific immunoglobulin E was associated with the incidence or severity of hypersensitivity adverse events. Serum and CSF ADA titers did not correlate with change in ML score. Therefore, the development of an ADA response to cerliponase alfa is not predictive of an adverse safety profile or poor treatment outcome.

Comparison of neutralizing antibody assays for receptor binding and enzyme activity of the enzyme replacement therapeutic Naglazyme (galsulfase).

Most patients receiving Naglazyme (galsulfase, rhASB) enzyme replacement therapy for mucopolysaccharidosis type VI develop an antibody response. To evaluate the impact of this response, two in vitro neutralizing antibody (NAb) assays were developed based on the two steps of the mechanism of action. Neutralization of enzyme activity was detected by inhibition of rhASB cleavage of a fluorogenic substrate. Neutralization of receptor binding was detected by decreased binding of labeled rhASB to immobilized soluble receptor. For the enzyme activity NAb assay, serum pretreatment was required to isolate antibodies from interfering phosphate ions, with sensitivity of < or =5 microg/mL. The receptor binding NAb assay used a five-fold dilution, with sensitivity of < or =40 microg/mL. Cutpoints for percent inhibition were based on 95% confidence intervals from naïve sera. Clinical samples were similarly likely to be positive in both assays than positive for neutralization of only one step in the mechanism of action. The two NAb assays yielded complementary information about potential neutralization of rhASB. Relative estimated sensitivity between neutralization assays did not correlate with the number of positive clinical samples or patients. In vitro NAb assays based on a well-understood mechanism of action provide specific information about the NAb mechanism.

Development, validation, and clinical implementation of an assay to measure total antibody response to naglazyme (galsulfase).

Naglazyme (galsulfase, rhASB) was developed as enzyme replacement therapy for mucopolysaccharidosis type VI. Naglazyme generated an IgG antibody response in most patients. To better characterize Naglazyme immunogenicity, a solution phase bridged immunoassay was developed to measure total antibody response regardless of isotype. Overnight incubation of serum dilutions with rhASB labeled with biotin and ruthenium-based tags allowed antibody-antigen complexes to form prior to capture on a streptavidin plate. Neat serum was tolerated in the assay, with a 1:10 screening dilution implemented for testing. At this dilution, the assay was sensitive to 75 ng/ml anti-rhASB. Titers were reported as the highest dilution factor with signal above a 95% confidence interval from naïve individual sera. Precise measurement of titers, within two consecutive dilution factors, was observed across analysts and days. Clinical samples showed similar positive/negative results between the IgG ELISA and the total antibody ECLA, although with an imperfect correlation. Improvements in assay performance and implementation strategy altered some positive clinical samples to negative and vice versa. Comparison of the titer readout for clinical samples with the screening signal illustrates a range of relationships for signal versus sample dilution factor, confirming that signal from a screening dilution cannot directly predict the reported titer.