Valoctocogene roxaparvovec is an adeno-associated virus serotype 5 (AAV5) based gene therapy vector causing the expression of the B-domain deleted SQ form of a recombinant human factor VIII (hFVIII-SQ) under the control of a liver-specific promoter. The expressed hFVIII-SQ replaces the missing coagulation factor VIII needed for effective haemostasis. Following valoctocogene roxaparvovec infusion, vector DNA is processed in vivo to form full-length, episomal transgenes that persist as the stable DNA forms that support long-term production of hFVIII-SQ.
Valoctocogene roxaparvovec transgene DNA levels (total amount of vector DNA) in various tissues (evaluated in nonclinical studies), blood, and shedding matrices were determined using a quantitative polymerase chain reaction (qPCR) assay. This assay is sensitive to transgene DNA, including fragments of degraded DNA. It does not indicate whether DNA is present in the vector capsid, in cells or in the fluid phase of the matrix (e.g., blood plasma, seminal fluid), or whether intact vector is present. Plasma and semen matrices were further evaluated by measuring encapsidated (potentially infectious) vector DNA using an immunoprecipitation quantitative PCR assay in studies 270-201 and 270-301.
Administration of valoctocogene roxaparvovec resulted in detectable vector DNA in blood and all shedding matrices evaluated, with peak concentrations observed between 1 and 9 days post-administration. The peak vector DNA concentrations were observed in blood, followed by saliva, semen, stool, and urine. The peak concentration observed to date in blood across studies 270-201 and 270-301 was 2 × 1011 vg/mL. The maximum concentration in any shedding matrix was 1 × 1010 vg/mL. After reaching the maximum in a matrix, the transgene DNA concentration declines steadily.
In the 141 evaluable patients from studies 270-201 and 270-301, encapsidated (potentially infectious) vector DNA was detectable in plasma up to 10 weeks after valoctocogene roxaparvovec administration.
In the 140 evaluable patients from studies 270-201 and 270-301, all patients achieved clearance of vector DNA in semen with a maximum time to clearance of 36 weeks. In the 138 evaluable patients from studies 270-201 and 270-301, the maximum time to clearance of encapsidated (potentially infectious) vector DNA in semen was 12 weeks.
In both studies, all patients cleared in urine and saliva, and 126 (89%) patients cleared in stool by the time of the data cut. The maximum time to clearance was 8 weeks for urine, 69 weeks for saliva, and 131 weeks for stool.
Magnitude and duration of shedding appear to be independent of the patient’s attained factor VIII activity.
No pharmacokinetic studies using valoctocogene roxaparvovec have been conducted in special populations.
A single intravenous administration of up to 2 × 1014 vg/kg of valoctocogene roxaparvovec in immunocompetent male mice with intact coagulation (CD1 mice), followed by an observation period of up to 26 weeks, showed dose-dependence of plasma levels of the expressed hFVIII-SQ protein and overall factor VIII activity in plasma. Transgene DNA was detected predominantly in spleen and liver, with lower DNA levels still detected at the end of the study (day 182) in lung, mesenteric lymph node, kidney, heart, testis and brain. Vector RNA transcripts were also detected predominantly in the liver with remaining low RNA levels in lung, heart, brain, kidney, lymph nodes, spleen and testis on study end (day 182).
There were no toxicities associated with valoctocogene roxaparvovec in CD1 mice observed for 26 weeks following single doses up to 2 × 1014 vg/kg, except a pattern of haemorrhages, necrosis and fibrosis, occurring primarily in the heart, lungs, epididymis and thymus that was consistent with a coagulopathy likely caused by the formation of antibodies directed against the expressed hFVIII-SQ, which also cross-reacted with the murine factor VIII protein.
In non-GLP studies conducted in NHP dosed up to 6 × 1013 vg/kg, an immune response specific for the AAV5 capsid, and an immune response specific to the heterologous hFVIII-SQ protein were observed, associated with transient APTT prolongation in a subset of non-human primates.
Vector integration was found following evaluation of liver samples in 12 non-human primates, collected up to 26 weeks following doses up to 6 × 1013 vg/kg of valoctocogene roxaparvovec (which is the corresponding dose level in humans) (see section 4.4 Risk of malignancy as a result of vector integration).
No carcinogenicity study was performed with valoctocogene roxaparvovec.
No dedicated reproductive and developmental toxicity studies, including embryo-foetal and fertility assessments, were performed with valoctocogene roxaparvovec, as males comprise the majority of the patient population to be treated with valoctocogene roxaparvovec. Since hFVIII-SQ DNA has been estimated to persist around/until 67 weeks in testes of CD1 mice after IV injection of a 6 × 1013 vg/kg dose, the potential for vertical transmission to offspring was studied in Rag2-/- mice. There were no instances of germline transmission to pups sired by male mice dosed with valoctocogene roxaparvovec, when assessing liver of F1 pups for hFVIII-SQ DNA by qPCR.
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