Key Points Nonacog beta pegol, a recombinant glycoPEGylated FIX with extended half-life, was developed to improve care for patients with hemophilia B. Weekly prophylaxis with nonacog beta pegol was well tolerated and was associated with low bleeding rates and an improved quality of life.
We previously demonstrated that rAAV vectors carrying human and canine factor IX (FIX) cDNA can infect, stably persist, and secrete functional human and canine FIX following direct intramuscular injection. In an attempt to improve FIX protein secretion for eventual therapeutic use, we set out to determine if alteration of the AAV capsid would affect skeletal muscle transduction and factor IX secretion. Two reasons to pursue this question were (1) the persistence of high-titer neutralizing antibody (NAB) to AAV2 and (2) our previous study that supported a restricted tropism of muscle fiber types to AAV2 transduction. Using an identical CMV/canine factor IX (cFIX) expression cassette, we cross-packaged this genome into virions generated from each of the five AAV serotypes. In a dose-response assay, equivalent amounts of rAAV/cFIX serotypes were tested in vitro and in vivo. In tissue culture cells, FIX antigen levels secreted into the supernatant varied depending on the AAV serotype used; type 2 transduced maximally, with serotypes 3, 1, 5, and 4, respectively, expressing lower levels. However, when the same viruses were tested in vivo using immunodeficient NOD/SCID animals, we obtained surprisingly different results. While the time to onset of detectable serum levels appeared the same for all serotypes, types 1, 3, and 5 produced 100- to 1000-fold more cFIX than type 2. In fact, 12 weeks after transduction, type 1 continued to express levels of cFIX on average at 80 microg/ml followed by type 5 (6.52 microg/ml), type 3 (3.27 microg/ml), type 4 (258 ng/ml), and finally type 2 (90 ng/ml). Coagulant activity of cFIX as measured by aPTT supported the circulating levels measured by ELISA demonstrating the secreted protein was functional, and RT-PCR of injected tissue correlated with the serotype-specific transduction data. In summary, we found significant differences in cFIX expression upon introducing various rAAV serotypes into mouse muscle. These data have direct bearing on the design of AAV gene therapy clinical trials for hemophilia and should also extend to most therapeutic transgenes.
Fanconi anemia (FA) is a genetic disorder characterized by bone marrow failure, congenital abnormalities, cancer susceptibility, and a marked cellular hypersensitivity to DNA interstrand cross-linking agents, which correlates with a defect in ability to repair this type of damage. We have previously identified an approximately 230-kDa protein present in a nuclear protein complex in normal human lymphoblastoid cells that is involved in repair of DNA interstrand cross-links and shows reduced levels in FA-A cell nuclei. The FANCA gene appears to play a role in the stability or expression of this protein. We now show that p230 is a well known structural protein, human ␣ spectrin II (␣SpII⌺*), and that levels of ␣SpII⌺* are not only significantly reduced in FA-A cells but also in FA-B, FA-C and FA-D cells (i.e. in all FA cell lines tested), suggesting a role for these FA proteins in the stability or expression of ␣SpII⌺*. These studies also show that ␣SpII⌺* forms a complex in the nucleus with the FANCA and FANCC proteins. ␣SpII⌺* may thus act as a scaffold to align or enhance interactions between FA proteins and proteins involved in DNA repair. These results suggest that FA represents a disorder in which there is a deficiency in ␣SpII⌺*.
The previously published mortality studies are limited in hemophilia populations but suggest that there is no increased risk of mortality in factor VIII inhibitor patients. This retrospective study analyzed surveillance data collected on 7,386 males with severe hemophilia A over a 13-year period to assess the association between a current inhibitor and death. During the study period, 432 participants died, among whom 48 were patients with an inhibitor. Clinical characteristics most strongly associated with death were increased number of reported bleeds, signs of liver disease, infection with either HIV or HCV, and the presence of inhibitor. Patients who underwent successful tolerization were not considered inhibitor patients in our analysis. In a multivariable analysis, the odds of death were 70% higher among patients with a current inhibitor compared to those without an inhibitor (P < 0.01). Deaths among patients with inhibitors were much more likely to be attributed to bleeding complications than those among patients without an inhibitor (42 vs. 12%, P < 0.0001). We conclude that males with severe hemophilia A and a current inhibitor are at increased risk of death.
Gene therapy has the potential to maintain therapeutic blood clotting factor IX (FIX) levels in patients with hemophilia B by delivering a functional human F9 gene into liver cells. This phase 1/2, open-label dose-escalation study investigated BAX 335 (AskBio009, AAV8.sc-TTR-FIXR338Lopt), an adeno-associated virus (AAV) serotype 8-based FIX Padua gene therapy, in patients with hemophilia B. This report focuses on 12-month interim analyses of safety, pharmacokinetics, effects on FIX activity, and immune responses for dosed participants. Eight adult males (\aged 20-69 years; range FIX activity, 0.5%-2.0%) received 1 of 3 BAX 335 IV doses: 2.0 × 1011; 1.0 × 1012; or 3.0 × 1012 vector genomes/kg. Three (37.5%) participants had 4 serious adverse events (SAEs), all considered unrelated to BAX 335. No SAE led to death. No clinical thrombosis, inhibitors, or other FIX Padua-directed immunity were reported. FIX expression was measurable in 7 of 8 participants; peak FIX activity displayed dose dependence (32.0%-58.5% in cohort 3). One participant achieved sustained therapeutic FIX activity of ~20%, without bleeding or replacement therapy, for 4 years; in others, FIX activity was not sustained beyond 5-11 weeks. In contrast to some previous studies, corticosteroid treatment did not stabilize FIX activity loss. We hypothesize that the loss of transgene expression could have been caused by stimulation of innate immune responses including CpG oligodeoxynucleotides introduced into the BAX 335 coding sequence by codon optimization. Registered at www.clinicaltrials.gov as NCT01687608.
Conventional gene therapy of hemophilia A relies on the transfer of factor VIII (FVIII; encoded by the F8 gene) cDNA. We carried out spliceosome-mediated RNA trans-splicing (SMaRT) to repair mutant FVIII mRNA. A pre-trans-splicing molecule (PTM) corrected endogenous FVIII mRNA in F8 knockout mice with the hemophilia A phenotype, producing sufficient functional FVIII to correct the hemophilia A phenotype. This is the first description of phenotypic correction of a genetic defect by RNA repair in a knockout animal model. Our results indicate the feasibility of using SMaRT to repair RNA for the treatment of genetic diseases.
Dihydroflavins reductively release iron rapidly and quantitatively from purified horse spleen or horse heart ferritin. The NAD(P)H:flavin oxidoreductase from Beneckea harveyi is used to generate a constant concentration of dihydroflavin permitting a continuous assay for complete iron release. Sepharose-linked dihydroflavins are not competent to release ferritin iron, demonstrating that the dihydroflavin must pass through the channels of the protein shell prior to iron reduction. Several experiments fail to show any specific flavin binding site, though dihydroflavins do display saturation kinetics with very high apparent Km's. The rates of iron release by a number of dihydroflavin analogues show that the electron transfer is significantly rate determining in iron release by dihydroriboflavin, while diffusion of the dihydroflavin through the protein channel is slow in the release of iron by dihydroFMN. The rate of iron release is also dependent on the initial content of iron, having a maximum at 1200 iron atoms per ferritin.
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