Adenovirus-Associated Virus Vector-Mediated Gene Transfer in Hemophilia B

Department of Haematology, University College London Cancer Institute, London, United Kingdom.
New England Journal of Medicine (Impact Factor: 55.87). 12/2011; 365(25):2357-65. DOI: 10.1056/NEJMoa1108046
Source: PubMed

ABSTRACT Hemophilia B, an X-linked disorder, is ideally suited for gene therapy. We investigated the use of a new gene therapy in patients with the disorder.
We infused a single dose of a serotype-8-pseudotyped, self-complementary adenovirus-associated virus (AAV) vector expressing a codon-optimized human factor IX (FIX) transgene (scAAV2/8-LP1-hFIXco) in a peripheral vein in six patients with severe hemophilia B (FIX activity, <1% of normal values). Study participants were enrolled sequentially in one of three cohorts (given a high, intermediate, or low dose of vector), with two participants in each group. Vector was administered without immunosuppressive therapy, and participants were followed for 6 to 16 months.
AAV-mediated expression of FIX at 2 to 11% of normal levels was observed in all participants. Four of the six discontinued FIX prophylaxis and remained free of spontaneous hemorrhage; in the other two, the interval between prophylactic injections was increased. Of the two participants who received the high dose of vector, one had a transient, asymptomatic elevation of serum aminotransferase levels, which was associated with the detection of AAV8-capsid-specific T cells in the peripheral blood; the other had a slight increase in liver-enzyme levels, the cause of which was less clear. Each of these two participants received a short course of glucocorticoid therapy, which rapidly normalized aminotransferase levels and maintained FIX levels in the range of 3 to 11% of normal values.
Peripheral-vein infusion of scAAV2/8-LP1-hFIXco resulted in FIX transgene expression at levels sufficient to improve the bleeding phenotype, with few side effects. Although immune-mediated clearance of AAV-transduced hepatocytes remains a concern, this process may be controlled with a short course of glucocorticoids without loss of transgene expression. (Funded by the Medical Research Council and others; number, NCT00979238.).

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Available from: Cecilia Rosales, Sep 26, 2015
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    • "Replacement therapies using recombinant coagulation proteins are expensive. More recently, FIX gene therapy trials for Hemophilia B patients have been successful (Nathwani et al. 2011), and this method is yet to be applied to FVIII and other coagulation proteins. A major problem faced in FIX gene therapy is the low level of protein expression, in part due to a different codon usage in the organism that produces the protein (Thomas et al. 2003). "
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    ABSTRACT: Blood coagulation occurs through a cascade of enzymes and cofactors that produces a fibrin clot, while otherwise maintaining haemostasis. The 11 human coagulation factors (FG, FII-FXIII) have been identified across all vertebrates, suggesting that they emerged with the first vertebrates around 500 Mya. Human FVIII, FIX and FXI are associated with thousands of disease-causing mutations. Here we evaluated the strength of selective pressures on the 14 genes coding for the 11 factors during vertebrate evolution, and compared these with human mutations in FVIII, FIX and FXI. Positive selection was identified for fibrinogen (FG), FIII, FVIII, FIX and FX in the mammalian Primates and Laurasiatheria and the Sauropsida (reptiles and birds). This showed that the coagulation system in vertebrates was under strong selective pressures, perhaps to adapt against blood-invading pathogens. The comparison of these results with disease-causing mutations reported in FVIII, FIX and FXI showed that the number of disease-causing mutations and the probability of positive selection were inversely related to each other. It was concluded that when a site was under positive selection, it was less likely to be associated with disease-causing mutations. In contrast, sites under negative selection were more likely to be associated with disease-causing mutations and be destabilizing. A residue-by-residue comparison of the FVIII, FIX and FXI sequence alignments confirmed this. This improved understanding of evolutionary changes in FVIII, FIX and FXI provided greater insight into disease-causing mutations, and better assessments of the codon sites that may be mutated in applications of gene therapy.
    Molecular Biology and Evolution 12/2014; 31(11). DOI:10.1093/molbev/msu248 · 9.11 Impact Factor
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    • "Using an AAV-mediated approach, we demonstrated a dosedependent 2-to 4-fold increase in hepatic SphK activity. Importantly, this approach did not alter the activity of SphK in other metabolically active tissues including skeletal muscle, kidney and pancreas, supporting the known liver-specific tropism of the AAV8 serotype [24] [25]. Interestingly , regardless of the level of overexpression achieved, hepatic TAG levels were reduced by 30–50%. "
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    ABSTRACT: Hepatic insulin resistance is a major risk factor for the development of type 2 diabetes and is associated with the accumulation of lipids, including diacylglycerol (DAG), triacylglycerols (TAG) and ceramide. There is evidence that enzymes involved in ceramide or sphingolipid metabolism may have a role in regulating concentrations of glycerolipids such as DAG and TAG. Here we have investigated the role of sphingosine kinase (SphK) in regulating hepatic lipid levels. We show that mice on a high-fat high-sucrose diet (HFHS) displayed glucose intolerance, elevated liver TAG and DAG, and a reduction in total hepatic SphK activity. Reduced SphK activity correlated with downregulation of SphK1, but not SphK2 expression, and was not associated with altered ceramide levels. The role of SphK1 was further investigated by overexpressing this isoform in the liver of mice in vivo. On a low-fat diet (LFD) mice overexpressing liver SphK1, displayed reduced hepatic TAG synthesis and total TAG levels, but with no change to DAG or ceramide. These mice also exhibited no change in gluconeogenesis, glycogenolysis or glucose tolerance. Similarly, overexpression of SphK1 had no effect on the pattern of endogenous glucose production determined during a glucose tolerance test. Under HFHS conditions, normalization of liver SphK activity to levels observed in LFD controls did not alter hepatic TAG concentrations. Furthermore, DAG, ceramide and glucose tolerance were also unaffected. In conclusion, our data suggest that SphK1 plays an important role in regulating TAG metabolism under LFD conditions. Copyright © 2014. Published by Elsevier B.V.
    Biochimica et Biophysica Acta (BBA) - Molecular and Cell Biology of Lipids 12/2014; 1851(2). DOI:10.1016/j.bbalip.2014.12.002 · 5.16 Impact Factor
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    • "One example is the clinical trial for hemophilia B using rAAV serotype 8 that has shown long-term expression of the transgene by the liver with significant clinical benefit (Nathwani et al., 2011). The market authorization by the European Medicine Agency in 2012 of Glybera, an rAAV1 vector for the treatment of LPLD patients, was a milestone in the field of gene therapy and prompted many pharmaceutical companies to move into this field (Bryant et al., 2013). "
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    ABSTRACT: Gene therapy approaches using recombinant adeno-associated virus serotype 2 (rAAV2) and serotype 8 (rAAV8) have achieved significant clinical benefits. The generation of rAAV Reference Standard Materials (RSM) is key to providing points of reference for particle titer, vector genome titer, and infectious titer for gene transfer vectors. Following the example of the rAAV2RSM, here we have generated and characterized a novel RSM based on rAAV serotype 8. Production of the rAAV8RSM was carried out using transient transfection and the purification was based on density gradient ultracentrifugation. The rAAV8RSM was distributed for characterization along with standard assay protocols to sixteen laboratories worldwide. Mean titers and 95% confidence intervals were determined for capsid particles (mean, 5.50x1011 pt/ml; CI, 4.26x1011 to 6.75x1011 pt/ml), vector genomes (mean, 5.75x1011 vg/ml; CI, 3.05x1011 to 1.09x1012 vg/ml), and infectious units (mean, 1.26x109 IU/ml; CI, 6.46x108 to 2.51x109 IU/ml). Notably, there was a significant degree of variation between institutions for each assay despite the relatively tight correlation of assay results within an institution. This outcome emphasizes the need to use RSMs to calibrate the titers of rAAV vectors in preclinical and clinical studies at a time where the field is maturing rapidly. The rAAV8RSM has been deposited at the American Type Culture Collection (VR-1816) and is available to the scientific community.
    Human Gene Therapy 10/2014; 25(11). DOI:10.1089/hum.2014.057 · 3.76 Impact Factor
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