Integration-deficient lentiviral vectors expressing codon-optimized R338L human FIX restore normal hemostasis in Hemophilia B mice.

Integration-deficient lentiviral vectors (IDLVs) have been shown to transduce a wide spectrum of target cells and organs in vitro and in vivo and to maintain long-term transgene expression in nondividing cells. However, epigenetic silencing of episomal vector genomes reduces IDLV transgene expression levels and renders these safe vectors less efficient. In this article, we describe for the first time a complete correction of factor IX (FIX) deficiency in hemophilia B mice by IDLVs carrying a novel, highly potent human FIX cDNA. A 50-fold increase in human FIX cDNA potency was achieved by combining two mechanistically independent yet synergistic strategies: (i) optimization of the human FIX cDNA codon usage to increase human FIX protein production per vector genome and (ii) generation of a highly catalytic mutant human FIX protein in which the arginine residue at position 338 was substituted with leucine. The enhanced human FIX activity was not associated with liver damage or with the formation of human FIX-directed inhibitory antibodies and rendered IDLV-treated FIX-knockout mice resistant to a challenging tail-clipping assay. A novel S1 nuclease-based B1-quantitative polymerase chain reaction assay showed low levels of IDLV integration in mouse liver. Overall, this study demonstrates that IDLVs carrying an improved human FIX cDNA safely and efficiently cure hemophilia B in a mouse model.

[Noise magnetic fields mitigates the inhibition of secretion function of primary human villous trophoblasts induced by 50 Hz magnetic fields].

OBJECTIVE: To explore the effects of 50 Hz sinusoidal magnetic fields (MF) on secretion function of primary human villous trophoblasts in vitro, and the interference effect of "noise" MF. METHODS: The trophoblasts were isolated from human villus by trypsin digestion and incubated in DMEM medium.Then the trophoblasts were exposed to 0.4 mT 50 Hz MF and/or "noise" MF respectively for different durations. Each exposure group was matched with one control group which was from the same villus and cultured with the same condition except the MF exposure. The concentrations of human chorionic gonadotropin (HCG) and progesterone in the culture medium were measured by immunofluorescence. Statistical significance of differences between means was determined by one way-ANOVA with P<0.05 considered significant. RESULT: 50 Hz MF inhibited the HCG and progesterone secretion significantly when exposure for 72 h (compared with control group, P<0.05). There was no significant change of HCG and progesterone secretion when trophoblasts were exposed to 0.4 mT "noise" MF within 72 h (compared with control group, P>0.05). However, by superimposing the "noise" MF, the inhibition of HCG and progesterone secretion of trophoblasts induced by 50 Hz MF was eliminated. CONCLUSION: The exposure to 50 Hz MF for long period could inhibit trophoblasts secreting HCG and progesterone, and the "noise" MF with the same intensity could eliminate the effects induced by 50 Hz MF.

A Retrospective Study of the Cytokine Profile Changes in Mice with FVIII Inhibitor Development After Adeno-Associated Virus-Mediated Gene Therapy in a Hemophilia A Mouse Model.

The development of inhibitory autoantibodies to the infused clotting factor VIII (FVIII) is a major complication for severe hemophilia A management. Novel therapy options for hemophilia have significantly progressed in the last decade, and a gene therapy cure for hemophilia is becoming a reality. However, mechanistic studies of FVIII autoantibodies (FVIII inhibitors) have lagged behind and remain a challenge for both protein replacement and gene therapy. FVIII inhibitor formation is assumed to be a classical T cell-dependent immune response in which cytokines/chemokines play an important role. The study of cytokine profile changes during FVIII inhibitor development may be helpful to understand the mechanism of inhibitor development and to explore potential novel approaches that will minimize the risk. After FVIII-/- mice were treated with intravenous administration of an adeno-associated virus 8 vector encoding human FVIII, FVIII expression peaked at week 2 (W2), and FVIII inhibitor was thoroughly developed at week 8 (W8). W8 plasma that showed positive FVIII inhibitor, and W2 samples with negative FVIII inhibitor (anti-FVIII[+]), were subjected to multiplex cytokines measurement. W8 and W2 samples were both negative for FVIII inhibitor (anti-FVIII[-]) as the control. In comparison to mice in the anti-FVIII(-) group, mice in the anti-FVIII(+) group exhibited significantly elevated pro-inflammatory cytokines of interleukin (IL)-1, IL-6, IL-12p40, monocyte chemoattractant protein-1, macrophage inflammatory protein (MIP)-1, MIP-2, and tumor necrosis factor alpha (TNF-α), especially at higher titers. The anti-inflammatory cytokine of transforming growth factor beta (TGF-ß) was decreased at W2 in both groups. Multivariate analysis of the risk factors for FVIII inhibitor development showed peak FVIII activity at W2. IL-6 and TNF-α at W8 were positively correlated with inhibitor formation, and negatively correlated with the age starting gene therapy. Collectively, the elevated monocyte derived pro-inflammatory cytokines/chemokines, together with the decreased anti-inflammatory cytokine of TGF-ß at an early time point, may contribute to the persistent inflammatory environment in favor of an immune response toward FVIII inhibitor development.

Cytokine production by CD4+ T cells specific for coagulation factor VIII in healthy subjects and haemophilia A patients.

Haemophilia A patients treated with human factor VIII (fVIII) may develop antibody (Ab) inhibitors to fVIII. FVIII-specific CD4(+) T cells are common in haemophilia A patients, but also in healthy subjects who do not have a sustained anti-fVIII Ab response. Here, we examined the fVIII-induced IFN gamma-, IL-4- and TGF-beta1-producing CD4(+) T blasts by culturing peripheral blood mononuclear cells (PBMC) from controls and patients with recombinant fVIII. FVIII exposure significantly increased IFN gamma- and IL-4-, but not TGF-beta1-producing CD4(+) T blasts in patients with inhibitors. Patients without inhibitors had fVIII-induced IFN gamma- and TGF-beta1-, but not IL-4-producing CD4(+) T blasts. Controls did not have IL-4-producing CD4(+) T blasts. However, controls whose PMBC proliferated in response to fVIII had fVIII-induced CD4(+) T blasts that produced IFN-gamma, the number of which correlated with the intensity of the proliferative response to fVIII of their PMBC, whereas controls whose PMBC did not proliferate to fVIII had predominantly fVIII-induced CD4(+) T blasts that produced TGF-beta1. The presence in controls and patients without inhibitors of fVIII-induced IFN-gamma-producing CD4(+) T cells, but not IL-4-producing CD4(+) T cells, which are abundant in inhibitor patients, suggests a role of Th1 cells in initiating the immune response to fVIII, and of Th2 cells in the development of strong inhibitor production. The polarized high ratios of Th3/Th1 and Th3/Th2 in controls and patients without inhibitors suggest that a preponderance ofTh3 cells in the response to fVIII may help to maintain tolerance to fVIII.

[Apoptosis-related gene expression of human villous trophoblasts exposed to 50 Hz magnetic field].

OBJECTIVE: To study apoptosis-related gene expression of human villous trophoblasts exposed to 50 Hz magnetic field and to investigate the possible mechanism of human reproductive health effects caused by 50 Hz magnetic field. METHODS: Cultured human villous trophoblasts were exposed to 50 Hz magnetic field at 0.4 mT for 6, 48, 72 hours. Gene expressions of Bcl-2, Bax, Caspase-3, p53 and Fas were analyzed using real-time reverse transcription polymerase chain reaction (RT-PCR) assay. RESULTS: Within 72 hours, the average fold change for each gene was near 1.00, and there was no significant difference on expression pattern in each gene between exposure and control groups (P > 0.05). CONCLUSION: 0.4 mT 50 Hz magnetic field does not affect the apoptosis-related gene expression of human villous trophoblasts in vitro.

Employing a gain-of-function factor IX variant R338L to advance the efficacy and safety of hemophilia B human gene therapy: preclinical evaluation supporting an ongoing adeno-associated virus clinical trial.

Vector capsid dose-dependent inflammation of transduced liver has limited the ability of adeno-associated virus (AAV) factor IX (FIX) gene therapy vectors to reliably convert severe to mild hemophilia B in human clinical trials. These trials also identified the need to understand AAV neutralizing antibodies and empty AAV capsids regarding their impact on clinical success. To address these safety concerns, we have used a scalable manufacturing process to produce GMP-grade AAV8 expressing the FIXR338L gain-of-function variant with minimal (<10%) empty capsid and have performed comprehensive dose-response, biodistribution, and safety evaluations in clinically relevant hemophilia models. The scAAV8.FIXR338L vector produced greater than 6-fold increased FIX specific activity compared with wild-type FIX and demonstrated linear dose responses from doses that produced 2-500% FIX activity, associated with dose-dependent hemostasis in a tail transection bleeding challenge. More importantly, using a bleeding model that closely mimics the clinical morbidity of hemophilic arthropathy, mice that received the scAAV8.FIXR338L vector developed minimal histopathological findings of synovitis after hemarthrosis, when compared with mice that received identical doses of wild-type FIX vector. Hemostatically normal mice (n=20) and hemophilic mice (n=88) developed no FIX antibodies after peripheral intravenous vector delivery. No CD8(+) T cell liver infiltrates were observed, despite the marked tropism of scAAV8.FIXR338L for the liver in a comprehensive biodistribution evaluation (n=60 animals). With respect to the role of empty capsids, we demonstrated that in vivo FIXR338L expression was not influenced by the presence of empty AAV particles, either in the presence or absence of various titers of AAV8-neutralizing antibodies. Necropsy of FIX(-/-) mice 8-10 months after vector delivery revealed no microvascular or macrovascular thrombosis in mice expressing FIXR338L (plasma FIX activity, 100-500%). These preclinical studies demonstrate a safety:efficacy profile supporting an ongoing phase 1/2 human clinical trial of the scAAV8.FIXR338L vector (designated BAX335).

[Abnormal shift of connexin 43 gap-junction protein induced by 50 Hz electromagnetic fields in Chinese hamster lung cells].

OBJECTIVE: To study the effects of extremely low frequency magnetic fields(ELF MF) on the amount and localization of connexin 43(Cx43) gap-junction protein in the Chinese hamster lung(CHL) cells, and to explore the mechanism of ELF MF suppression on gap-junctional intercellular communication(GJIC). METHODS: The cells were irradiated for 24 h with 50 Hz sinusoidal magnetic field at 0.8 mT without or with 12-O-tetrade-canoylphorbol-3-acetate(TPA), 5 ng/ml for 1 h. The localization of Cx43 proteins were performed by indirect immunofluorescence histochemical analysis and detected by confocal microscopy. The second experiment was conducted to examine the quantity of Cx43 proteins level in nuclei or cytoplasm and detected by Western blotting analysis. RESULTS: The cells exposed to TPA for 1 h displayed less bright labelled spots in the regions of intercellular junction than the normal cells. Most of Cx43 labelled spots occurred in the cytoplasm and aggregated near the nuclei. At the same time, the amount of Cx43 protein in cytoplasm were increased[(2.03 +/- 0.89) in ELF group, (2.43 +/- 0.82) in TPA group] as compared to normal control(1.04 +/- 0.17) (P < 0.01). CONCLUSION: Inhibition on GJIC function by ELF MF alone or combined with TPA may be related with the shift of Cx43 from the regions of intercellular junction to the cytoplasm.