Adeno-associated virus (AAV) vectors are a leading platform for gene-based therapies for both monogenic and complex acquired disorders. The success of AAV gene transfer highlights the need to answer ...outstanding clinical questions of safety, durability, and the nature of the human immune response to AAV vectors. Here, we present longitudinal follow-up data of subjects who participated in the first trial of a systemically delivered AAV vector. Adult males (n = 7) with severe hemophilia B received an AAV2 vector at doses ranging from 8 × 1010 to 2 × 1012 vg/kg to target hepatocyte-specific expression of coagulation factor IX; a subset (n = 4) was followed for 12–15 years post-vector administration. No major safety concerns were observed. There was no evidence of sustained hepatic toxicity or development of hepatocellular carcinoma as assessed by liver transaminase values, serum α-fetoprotein, and liver ultrasound. Subjects demonstrated persistent, increased AAV neutralizing antibodies (NAbs) to the infused AAV serotype 2 (AAV2) as well as all other AAV serotypes tested (AAV5 and AAV8) for the duration of follow-up. These data represent the longest available longitudinal follow-up data of subjects who received intravascular AAV and support the preliminary safety of intravascular AAV administration at the doses tested in adults. Data demonstrate, for the first time, the persistence of high-titer, multi-serotype cross-reactive AAV NAbs for up to 15 years post- AAV vector administration. Our observations are broadly applicable to the development of AAV-mediated gene therapy.
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Presented here are the longest available longitudinal follow-ups of human subjects following intravascular adeno-associated virus (AAV) vector administration. These data support the preliminary long-term safety of systemic AAV delivery and demonstrate the persistence of high-titer, multi-serotype, cross-reactive AAV NAbs for up to 15 years after vector infusion.
Fitusiran is an RNA interference therapeutic that targets antithrombin (AT) in the liver and interferes with AT translation by binding and degrading messenger RNA-AT, thereby silencing AT gene ...expression and preventing AT synthesis. In both preclinical and clinical studies, AT knockdown results in dose-dependent AT lowering when fitusiran is given weekly or monthly subcutaneously. In clinical trials, fitusiran dose escalation has resulted in improved thrombin generation and clinical hemostasis as measured by reduction in annualized bleed rate. Unlike currently licensed drugs, this improvement was not only in patients with hemophilia A but in also those with hemophilia B, with or without inhibitors. In dental and surgical procedures, fitusiran also provided perioperative hemostasis in association with AT lowering. Fitusiran is well tolerated, with minor local injection site reactions, but in one subject with severe hemophilia A, the concomitant use of daily high-dose factor VIII, inconsistent with trial guidance to avoid high, repeat doses of clotting factor, was associated with fatal thrombosis, suggesting the need for caution when using hemostatic agents in conjunction with fitusiran. Preclinical in vitro and in silico studies indicate improvement in thrombin generation in rare bleeding disorder plasmas, including in plasmas from patients with severe factors V, VII, and X deficiency, suggesting potential therapeutic benefit.
This phase 3 pivotal study evaluated the safety, efficacy, and pharmacokinetics of a recombinant FVIII Fc fusion protein (rFVIIIFc) for prophylaxis, treatment of acute bleeding, and perioperative ...hemostatic control in 165 previously treated males aged ≥12 years with severe hemophilia A. The study had 3 treatment arms: arm 1, individualized prophylaxis (25-65 IU/kg every 3-5 days, n = 118); arm 2, weekly prophylaxis (65 IU/kg, n = 24); and arm 3, episodic treatment (10-50 IU/kg, n = 23). A subgroup compared recombinant FVIII (rFVIII) and rFVIIIFc pharmacokinetics. End points included annualized bleeding rate (ABR), inhibitor development, and adverse events. The terminal half-life of rFVIIIFc (19.0 hours) was extended 1.5-fold vs rFVIII (12.4 hours; P < .001). Median ABRs observed in arms 1, 2, and 3 were 1.6, 3.6, and 33.6, respectively. In arm 1, the median weekly dose was 77.9 IU/kg; approximately 30% of subjects achieved a 5-day dosing interval (last 3 months on study). Across arms, 87.3% of bleeding episodes resolved with 1 injection. Adverse events were consistent with those expected in this population; no subjects developed inhibitors. rFVIIIFc was well-tolerated, had a prolonged half-life compared with rFVIII, and resulted in low ABRs when dosed prophylactically 1 to 2 times per week. This trial was registered at www.clinicaltrials.gov as #NCT01181128.
Key Points
Prophylactic factor replacement in patients with hemophilia B improves outcomes but requires frequent injections. A recombinant factor IX Fc fusion protein (rFIXFc) with a prolonged half-life was ...developed to reduce the frequency of injections required.
We conducted a phase 3, nonrandomized, open-label study of the safety, efficacy, and pharmacokinetics of rFIXFc for prophylaxis, treatment of bleeding, and perioperative hemostasis in 123 previously treated male patients. All participants were 12 years of age or older and had severe hemophilia B (endogenous factor IX level of ≤2 IU per deciliter, or ≤2% of normal levels). The study included four treatment groups: group 1 received weekly dose-adjusted prophylaxis (50 IU of rFIXFc per kilogram of body weight to start), group 2 received interval-adjusted prophylaxis (100 IU per kilogram every 10 days to start), group 3 received treatment as needed for bleeding episodes (20 to 100 IU per kilogram), and group 4 received treatment in the perioperative period. A subgroup of group 1 underwent comparative sequential pharmacokinetic assessments of recombinant factor IX and rFIXFc. The primary efficacy end point was the annualized bleeding rate, and safety end points included the development of inhibitors and adverse events.
As compared with recombinant factor IX, rFIXFc exhibited a prolonged terminal half-life (82.1 hours) (P<0.001). The median annualized bleeding rates in groups 1, 2, and 3 were 3.0, 1.4, and 17.7, respectively. In group 2, 53.8% of participants had dosing intervals of 14 days or more during the last 3 months of the study. In groups 1, 2 and 3, 90.4% of bleeding episodes resolved after one injection. Hemostasis was rated as excellent or good during all major surgeries. No inhibitors were detected in any participants receiving rFIXFc; in groups 1, 2, and 3, 73.9% of participants had at least one adverse event, and serious adverse events occurred in 10.9% of participants. These events were mostly consistent with those expected in the general population of patients with hemophilia.
Prophylactic rFIXFc, administered every 1 to 2 weeks, resulted in low annualized bleeding rates in patients with hemophilia B. (Funded by Biogen Idec; ClinicalTrials.gov number, NCT01027364.).
Current factor VIII (FVIII) products display a half-life (t1/2) of ∼ 8-12 hours, requiring frequent intravenous injections for prophylaxis and treatment of patients with hemophilia A. rFVIIIFc is a ...recombinant fusion protein composed of a single molecule of FVIII covalently linked to the Fc domain of human IgG1 to extend circulating rFVIII t1/2. This first-in-human study in previously treated subjects with severe hemophilia A investigated safety and pharmacokinetics of rFVIIIFc. Sixteen subjects received a single dose of rFVIII at 25 or 65 IU/kg followed by an equal dose of rFVIIIFc. Most adverse events were unrelated to study drug. None of the study subjects developed anti-rFVIIIFc antibodies or inhibitors. Across dose levels, compared with rFVIII, rFVIIIFc showed 1.54- to 1.70-fold longer elimination t1/2, 1.49- to 1.56-fold lower clearance, and 1.48- to 1.56-fold higher total systemic exposure. rFVIII and rFVIIIFc had comparable dose-dependent peak plasma concentrations and recoveries. Time to 1% FVIII activity above baseline was ∼ 1.53- to 1.68-fold longer than rFVIII across dose levels. Each subject showed prolonged exposure to rFVIIIFc relative to rFVIII. Thus, rFVIIIFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia A. This trial was registered at www.clinicaltrials.gov as NCT01027377.
Gene therapy provides a potential phenotypic cure for hemophilia, yet the cost of this novel treatment is high, tempering enthusiasm and raising questions regarding cost vs benefit. To evaluate the ...cost-effectiveness of gene therapy treatment of severe hemophilia A compared with prophylaxis with factor VIII (FVIII), we developed a Markov state–transition model to estimate the costs and effectiveness of severe hemophilia A treatment strategies from a United States health care system perspective. Quality-adjusted life-years (QALYs) were the effectiveness measure. In the base case, hypothetical cohorts of 30-year-old patients received gene therapy or FVIII prophylaxis. We obtained model probabilities and utilities from the literature and costs from Medicare reimbursement data. One-way and probabilistic sensitivity analyses were performed to test the robustness of results. Over a 10-year time horizon, total per-person gene therapy strategy costs were $1.0M and resulted in 8.33 QALYs, whereas prophylaxis cost $1.7M and resulted in 6.62 QALYs. Thus, gene therapy dominated prophylaxis (costs less and was more effective). Gene therapy remained dominant unless initial costs exceeded $1.6M and were <$100 000 per 1 QALY gained compared with prophylaxis if initial costs were <$1.7M. Results were not sensitive to variation of all other parameters over clinically plausible ranges. In a probabilistic sensitivity analysis simultaneously varying all parameters 3000 times over parameter distributions, gene therapy was dominant in 92% of model iterations. Treatment of severe hemophilia A with gene therapy is likely to be cost-saving or cost-effective compared with FVIII prophylaxis.
•Gene therapy is cost-effective in severe hemophilia A compared with standard factor VIII prophylaxis.•Over a 10-year time horizon, gene therapy cost $1M and resulted in 8.33 QALYs gained, whereas prophylaxis cost $1.7M and resulted in 6.62 QALYs gained.
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Almost half of severe hemophilia A (HA) is caused by an intron 22 inversion mutation (Int22Inv), which disrupts the 26-exon
gene. Inverted
mRNA exons 1-22 are transcribed, while
mRNA, containing
...exons 23-26, is transcribed from a promoter within intron 22. Neither FVIII activity nor FVIII antigen (cross-reacting material, CRM) are detectable in plasma of patients with an intron-22 inversion.
To test the hypothesis that (putative) intracellular synthesis of FVIII proteins encoded by inverted
and
mRNAs confers T-cell tolerance to almost the entire FVIII sequence, and to evaluate the immunogenicity of the region encoded by the
exon 22-23 junction sequence.
Peripheral blood mononuclear cells (PBMCs) from 30 severe or moderate HA subjects (17 with an Int22Inv mutation) were tested by ELISPOT assays to detect cytokine secretion in response to FVIII proteins and peptides and to map immunodominant T-cell epitopes. Potential immunogenicity of FVIII sequences encoded by the
exon 22-23 junction region was also tested using peptide-MHCII binding assays.
Eight of the Int22Inv subjects showed robust cytokine secretion from PBMCs stimulated with FVIII proteins and/or peptides, consistent with earlier publications from the Conti-Fine group. Peptide ELISPOT assays identified immunogenic regions of FVIII. Specificity for sequences encoded within
mRNA exons 1-22 and
mRNA was confirmed by staining Int22Inv CD4
T cells with peptide-loaded HLA-Class II tetramers. FVIII peptides spanning the
exon 22-23 junction (encoding M2124-V2125) showed limited binding to MHCII proteins and low immunogenicity, with cytokine secretion from only one Int22Inv subject.
PBMCs from multiple subjects with an Int22Inv mutation, with and without a current FVIII inhibitor, responded to FVIII epitopes. Furthermore, the FVIII region encoded by the exon 22-23 junction sequence was not remarkably immunoreactive and is therefore unlikely to contain an immunodominant, promiscuous CD4
T-cell epitope. Our results indicate that putative intracellular expression of partial FVIII proteins does not confer T-cell tolerance to FVIII regions encoded by inverted
mRNA or
mRNA.
Current factor IX (FIX) products display a half-life (t1/2) of ∼ 18 hours, requiring frequent intravenous infusions for prophylaxis and treatment in patients with hemophilia B. This open-label, ...dose-escalation trial in previously treated adult subjects with hemophilia B examined the safety and pharmacokinetics of rFIXFc. rFIXFc is a recombinant fusion protein composed of FIX and the Fc domain of human IgG1, to extend circulating time. Fourteen subjects received a single dose of rFIXFc; 1 subject each received 1, 5, 12.5, or 25 IU/kg, and 5 subjects each received 50 or 100 IU/kg. rFIXFc was well tolerated, and most adverse events were mild or moderate in intensity. No inhibitors were detected in any subject. Dose-proportional increases in rFIXFc activity and Ag exposure were observed. With baseline subtraction, mean activity terminal t1/2 and mean residence time for rFIXFc were 56.7 and 71.8 hours, respectively. This is ∼ 3-fold longer than that reported for current rFIX products. The incremental recovery of rFIXFc was 0.93 IU/dL per IU/kg, similar to plasma-derived FIX. These results show that rFIXFc may offer a viable therapeutic approach to achieve prolonged hemostatic protection and less frequent dosing in patients with hemophilia B. The trial was registered at www.clinicaltrials.gov as NCT00716716.
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