Etranacogene dezaparvovec is a gene therapy product designed to introduce a copy of the human Factor IX coding DNA sequence into hepatocytes to address the root cause of the Haemophilia B disease. Etranacogene dezaparvovec consists of a codon-optimised coding DNA sequence of the gain-of-function Padua variant of the human Factor IX (hFIXco-Padua), under control of the liver-specific LP1 promoter, encapsulated in a non-replicating recombinant adeno-associated viral vector of serotype 5 (AAV5).
Following single intravenous infusion, etranacogene dezaparvovec preferentially targets liver cells, where the vector DNA resides almost exclusively in episomal form. After transduction, etranacogene dezaparvovec directs long-term liver-specific expression of Factor IX-Padua protein. As a result, etranacogene dezaparvovec partially or completely ameliorates the deficiency of circulating Factor IX procoagulant activity in patients with Haemophilia B.
The etranacogene dezaparvovec-derived Factor IX protein produced in the liver is expected to undergo similar distribution and catabolic pathways as the endogenous native Factor IX protein in people without Factor IX deficiency.
The pharmacokinetics of shedding was characterised following etranacogene dezaparvovec administration, using a sensitive polymerase chain reaction (PCR) assay to detect vector DNA sequences in blood and semen samples, respectively. 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.
In the phase 3 study, detectable vector DNA with a maximum vector DNA concentrations post-dose was observed in blood (n = 53/54) and semen (n = 42/54) at a median time (Tmax) of 4 hours and 42 days, respectively. The mean peak concentrations were 2.2 × 1010 copies/mL and 3.8 × 105 copies/mL in blood and semen, respectively. After reaching the maximum in a matrix, the transgene DNA concentration declines steadily. Shedding-negative status in patients was defined as having 3 consecutive samples at vector DNA concentration below the limit of detection (<LOD). Using this definition, a total of 56% (30/54) of patients reached absence of vector DNA from blood and 69% (37/54) from semen by month 24. The median time to absence of shedding was 52.3 weeks in blood and 45.8 weeks in semen at 24 months post-dose. Several subjects did not return the required number of blood and semen samples to assess the shedding status as per the definition. Considering shedding results obtained from the final 2 available consecutive samples, a total of 40/54 (74%) and 47/54 (87%) patients were identified to have reached absence of vector DNA from blood and semen, respectively, at 24 months post-dose.
In the phase 3 study, majority (n=45) of the patients had normal renal function (creatinine clearance (CLcr) = ≥90 mL/min defined by Cockcroft-Gault equation), 7 patients had mild renal impairment (CLcr = 60 to 89 mL/min) and 1 patient had moderate renal impairment (CLcr = 30 to 59 mL/min). No clinically relevant differences in Factor IX activity were observed between these patients.
Etranacogene dezaparvovec was not studied in patients with severe renal impairment (CLcr = 15 to 29 mL/min) or end-stage renal disease (CLCr <15 mL/min).
In the phase 3 study, patients with varying degree of liver steatosis at baseline showed no clinically relevant different Factor IX activity levels.
Patients with severe liver impairment and advanced fibrosis were not studied.
Preclinical studies were initiated with a gene therapy product employing the recombinant adenoassociated virus serotype 5 (rAAV5) expressing the wild type of the human coagulation Factor IX (rAAV5-hFIX). Etranacogene dezaparvovec (rAAV5-hFIX-Padua) was subsequently developed from rAAV5-hFIX by introduction of a 2 nucleotide change in the transgene for human Factor IX, generating thereby the naturally occurring Padua variant of Factor IX, which exhibits significantly augmented activity.
The No Observed-Adverse-Effect-Level (NOAEL) was observed at 9 × 1013 gc/kg body weight in nonhuman primates, which is approximately 5-fold above the human etranacogene dezaparvovec dose of 2 × 1013 gc/kg body weight.
Biodistribution of etranacogene dezaparvovec and its predecessor, the gene therapy of human wild type Factor IX, was assessed in mice and non-human primates following intravenous administration. Dose-dependent preferential distribution to the liver was confirmed for both vectors and their transgene expression.
Genotoxic and reproductive risks were evaluated with the rAAV5-hFIX. The integration site analysis in host genomic DNA was performed on liver tissue from mice and non-human primates injected with rAAV5-hFIX up to a dose of 2.3 × 1014 gc/kg body weight, corresponding to approximately 10-fold higher than the clinical dose in human. The retrieved rAAV5-hFIX vector DNA sequences represented almost exclusively episomal forms that were non-integrated into the host DNA. The remaining low level of integrated rAAV5-hFIX DNA was distributed throughout the host genome with no preferred integration in genes associated with mediation of malignant transformation in human.
No dedicated carcinogenicity studies were performed with etranacogene dezaparvovec. Although there are no fully adequate animal models to address the tumorigenic and carcinogenic potential of etranacogene dezaparvovec in human, toxicological data do not suggest concern for tumourigenicity.
No dedicated reproductive and developmental toxicity studies, including embryo foetal and fertility assessments, were performed with etranacogene dezaparvovec, as males comprise the majority of the patient population to be treated with etranacogene dezaparvovec. The risk of germline transmission after administration of 2.3 × 1014 gc/kg body weight rAAV5-hFIX, i.e. a dose approximately 10-fold higher than recommended for humans, was assessed in mice. The rAAV5-hFIX administration resulted in detectable vector DNA in the reproductive organs and sperm of male animals. However, following mating of these mice with naïve female animals at 6 days after administration, the rAAV5-hFIX vector DNA was not detected in the female reproductive tissues nor offspring, indicating no paternal germline transmission.
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