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


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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    • "In recent years, next-generation biomolecule formats such as viral vectors for gene therapy and oncolytic viruses for virotherapy have been gaining increasing importance. The first gene therapy product is already approved for sale in Europe to compensate for lipoprotein lipase deficiency (uniQure, 2012) and further recombinant adenoassociated virus (rAAV) based therapies, e.g., for treatment of eye and blood coagulation related disorders seem very promising in clinical trials (MacLaren et al., 2014; Maguire et al., 2009; Nathwani et al., 2011). Hence, the demand for economic large-scale manufacturing processes of viral vectors is raised, especially of those that are based on rAAV. "
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    ABSTRACT: Cell engineering and bioprocess optimizations such as low temperature cultivation represent powerful tools to improve cellular performance and product yields of mammalian production cells. Besides monoclonal antibodies (mABs), novel biotherapeutic formats such as viral vectors will gain increasing importance. Here, we demonstrate that similar to Chinese hamster ovary (CHO) cells, product yields of recombinant adeno-associated virus (rAAV) producing HeLa cells can be markedly increased by low temperature cultivation. MicroRNAs (miRNAs) are small non-coding RNAs that critically regulate cell phenotypes. We thus investigated differential miRNA expression in response to mild hypothermia in CHO and HeLa production cells. We discovered miR-483 to be substantially up-regulated upon temperature down-shift in both cell types. Functional validation experiments revealed that introduction of miR-483 mimics led to a significant increase in both rAAV and mAB production in HeLa and CHO cells, respectively. Furthermore, inhibition of miR-483 up-regulation during mild hypothermia significantly decreased product yields, suggesting that miR-483 is a key regulator of cellular productivity in mammalian cells. In addition, miRNA target gene identification indicated that miR-483 might regulate genes directly involved in cellular survival and protein expression. Our results highlight that miR-483 is a valuable tool for product-independent engineering of mammalian production cells. This article is protected by copyright. All rights reserved.
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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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