Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases

Letter to the Edito

Muscle Specific and Efficient Transgene Expression Using the Fast and Slow Troponin I Promoters in Cell Culture

NRC publication: Ye

Differential effects of dystrophin and utrophin gene transfer in immunocompetent muscular dystrophy (mdx) mice

Duchenne muscular dystrophy (DMD) is a fatal disease caused by defects in the gene encoding dystrophin. Dystrophin is a cytoskeletal protein, which together with its associated protein complex, helps to protect the sarcolemma from mechanical stresses associated with muscle contraction. Gene therapy efforts aimed at supplying a normal dystrophin gene to DMD muscles could be hampered by host immune system recognition of dystrophin as a "foreign" protein. In contrast, a closely related protein called utrophin is not foreign to DMD patients and is able to compensate for dystrophin deficiency when overexpressed throughout development in transgenic mice. However, the issue of which of the two candidate molecules is superior for DMD therapy has remained an open question. In this study, dystrophin and utrophin gene transfer effects on dystrophic muscle function were directly compared in the murine (mdx) model of DMD using E1/E3-deleted adenovirus vectors containing either a dystrophin (AdV-Dys) or a utrophin (AdV-Utr) transgene. In immunologically immature neonatal animals, AdV-Dys and AdV-Utr improved tibialis anterior muscle histopathology, force-generating capacity, and the ability to resist injury caused by high-stress contractions to an equivalent degree. By contrast, only AdV-Utr was able to achieve significant improvement in force generation and the ability to resist stress-induced injury in the soleus muscle of immunocompetent mature mdx animals. In addition, in mature mdx mice, there was significantly greater transgene persistence and reduced inflammation with utrophin compared to dystrophin gene transfer. We conclude that dystrophin and utrophin are largely equivalent in their intrinsic abilities to prevent the development of muscle necrosis and weakness when expressed in neonatal mdx animals with an immature immune system. However, because immunity against dystrophin places an important limitation on the efficacy of dystrophin gene replacement in an immunocompetent mature host, the use of utrophin as an alternative to dystrophin gene transfer in this setting appears to offer a significant therapeutic advantage.NRC publication: Ye

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route

NRC publication: Ye

Process intensification for high yield production of influenza H1N1 Gag virus-like particles using an inducible HEK-293 stable cell line

Influenza virus dominant antigens presentation using virus like particle (VLP) approach is attractive for the development of new generation of influenza vaccines. Mammalian cell platform offers many advantages for VLP production. However, limited attention has been paid to the processing of mammalian cell produced VLPs. Better understanding of the production system could contribute to increasing the yields and making large-scale VLP vaccine manufacturing feasible. In a previous study, we have generated a human embryonic kidney HEK-293 inducible cell line expressing Hemagglutinin (HA) and Neuraminidase (NA), which was used to produce VLPs upon transient transfection with a plasmid containing HIV-1 Gag. In this work, to streamline the production process, we have developed a new HEK-293 inducible cell line adapted to suspension growth expressing the three proteins HA, NA (H1N1 A/PR/8/1934) and the Gag fused to GFP for monitoring the VLP production. The process was optimized to reach higher volumetric yield of VLPs by increasing the cell density at the time of induction without sacrificing the cell specific productivity. A 5-fold improvement was achieved by doing media evaluation at small scale. Furthermore, a 3-L perfusion bioreactor mirrored the performance of small-scale shake flask cultures with sequential medium replacement. The cell density was increased to 14 7 106 cells/ml at the time of induction which augmented by 60-fold the volumetric yield to 1.54 7 1010 Gag-GFP fluorescent events/ml, as measured by flow cytometry. The 9.5-L harvest from the perfusion bioreactor was concentrated by tangential flow filtration at low shear rate. The electron micrographs revealed the presence of VLPs of 100\u2013150 nm with the characteristic dense core of HIV-1 particles. The developed process shows the feasibility of producing high quantity of influenza VLPs from an inducible mammalian stable cell line aiming at large scale vaccine manufacturing.Peer reviewed: YesNRC publication: Ye

Modulation of Starling forces and muscle fiber maturity permits adenovirus-mediated gene transfer to adult dystrophic (mdx) mice by the intravascular route

NRC publication: Ye

High-Level Recombinant Protein Production in CHO Cells Using an Adenoviral Vector and the Cumate Gene-Switch

DA - 20070202IS - 8756-7938 (Print)LA - ENGPT - JOURNAL ARTICLENRC publication: Ye