Fidanacogene elaparvovec is a gene therapy designed to introduce a functional copy of the high activity Padua variant of the factor IX gene (FIX-R338L) in the transduced cells to address the monogenic root cause of haemophilia B.
Fidanacogene elaparvovec is a non-replicating recombinant AAV vector that utilises AAVRh74var capsid to deliver a stable human factor IX transgene. AAVRh74var capsid is able to transduce hepatocytes, the natural site of factor IX synthesis. The factor IX gene present in fidanacogene elaparvovec is designed to reside predominately as episomal DNA within transduced cells and expression of the transgene is driven by a liver specific promoter, which results in tissue specific, continuous and sustained factor IX protein expression.
Fidanacogene elaparvovec therapy results in measurable vector-derived coagulation factor IX activity.
Fidanacogene elaparvovec vector DNA levels were measured and quantified in blood and various shedding matrices using a quantitative polymerase chain reaction (qPCR) assay. This assay is sensitive and specific to fidanacogene elaparvovec vector DNA, but could also detect DNA fragments.
Vector shedding after infusion with fidanacogene elaparvovec was assessed in 60 patients at multiple time points in clinical studies (C0371005/C0371003 and C0371002). Vector DNA was shed in peripheral blood mononuclear cells (PBMC), saliva, urine, semen, and serum/plasma. In general, peak levels of vector DNA occurred within the first two weeks after infusion. Highest peak vector DNA concentrations were found in serum/plasma compared to the other liquid matrices (saliva, urine, semen). In plasma (measured only in C0371002), mean peak vector DNA concentration of 2.008 × 109 vg/mL was observed. The mean peak vector DNA concentration in any shedding matrix was 6.261 × 106 vg/mL.
Full clearance of vector DNA was defined as having 3 consecutive negative results (i.e., below quantification limit; BQL). Vector DNA fully cleared in serum, plasma, saliva, and semen within a mean of 1-4 months after infusion and PBMC was slowest fluid to full clearance within a mean of 12 months. In urine, the peak vector DNA concentration was very low relative to plasma and declined to full clearance within a mean of 4 weeks after infusion. Across studies, the maximum observed time for vector DNA full clearance in saliva, urine and semen were 105 days, 87 days and 154 days, respectively.
To further characterise the shed material, saliva, semen, and urine samples from a subset of 17 patients in Study C0371002 were tested using nuclease treatment (MNase) prior to DNA extraction. Nuclease treatment digests the free floating vector DNA so it cannot be quantified, ensuring the material being quantified following digestion is only encapsulated viral DNA. After nuclease treatment and subsequent DNA extraction, the amount of fidanacogene elaparvovec was measured by qPCR. In saliva, mean concentrations were similar up to week 2 between the MNase treatment and without MNase treatment subgroups, while all participants had concentrations BQL by week 9. In semen, mean concentrations were approximately 33% lower in the MNase treatment subgroup until week 3, and BQL for all participants by week 11. In urine, mean concentrations were approximately 30% lower in the MNase treatment subgroup until 72 h post-infusion and were BQL for all participants by week 2.
No adverse findings were observed in a 90-day single-dose intravenous general toxicity study in cynomolgus monkeys at doses up to 5 × 1012 vg/kg (10 times the recommended human dose). In a monkey biodistribution study, 22 tissues were collected 30 and 92 days following treatment. The highest levels of vector DNA were found in liver with levels approximately 20-fold higher than spleen, the organ with second most abundant levels of genomic DNA. There was very little biodistribution to testes.
In a 2-year vector integration study in cynomolgus monkeys administered 5 × 1012 vg/kg (10 times the recommended human dose), there was no indication that integration of vector DNA into host cell DNA resulted in altered liver function, or hepatocellular hyperplasia and carcinoma up to 2 years. The integration profile was considered benign as the integrations were generally random with a low frequency that was below published spontaneous mutation rate estimates for the liver and due to the absence of significant clonal expansion. Nonclinical safety data available beyond 2 years has not been established.
Carcinogenicity studies have not been conducted. The results of the integration site analysis conducted in cynomolgus monkeys and haemophilia B dogs indicated a benign profile and there was no evidence of clonal expansion. There was no evidence of hepatocellular hyperplasia in monkeys at the 92-day or 2-year necropsied, nor in mice in the 1-year study.
No dedicated reproductive and developmental toxicity studies, including embryofoetal and fertility assessments, were performed with fidanacogene elaparvovec, as males comprise the majority of the patient population to be treated with fidanacogene elaparvovec. The potential for germline transmission has been evaluated in male rabbits and vector was no longer detectable in semen at 5 months post-administration.
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