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Abstract No. 156
The HyVec System: High-Capacity Adenovirus/Lentivirus Vectors Achieve Efficient Two-Stage Gene Transfer and Stable Integration of Transgenes
Lily J.-L. Jih,1 Shuji Kubo,2 Celina Ngiam,1 Maria Barcova,1 Emmanuelle Faure,1 Ji Hee Kim,1 Toshiaki Shichinohe,1,3 Ataru Sazawa,1,3 Ruth Margalit,1 Renata Stripecke,1,3 Paula Cannon,1,4 Noriyuki Kasahara,1,3 Kohnosuke Mitani.2
1Vector Development Group, IntraGene Sciences, Inc., Los Angeles, CA, United States;
2Microbiology and Immunology, University of California Los Angeles, Los Angeles, CA, United States;
3Institute for Genetic Medicine, University of Southern California, Los Angeles, CA, United States;
4Microbiology and Immunology, Uniformed Services University of the Health Sciences, Bethesda, MD, United States
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We have successfully generated and tested a novel hybrid vector system that utilizes high-capacity helper-dependent adenovirus as a carrier to deliver sequences encoding either HIV-based lentivirus or MLV-based retrovirus vectors. This adenovirus-retro/lentivirus hybrid vector (HyVec) system thus achieves efficient and permanent transduction via a two-stage mechanism: the retrovirus or lentivirus elements carried by the adenovirus are transiently produced in transduced cells during the first (adenovirus) stage, followed by gene transfer and stable integration in additional adjacent cells via the second (retro/lentivirus) stage.[br]For proof-of-principle, the MLV vector used in our first-generation adenovirus-retrovirus hybrid carried a GFP marker gene, is ecotropic (ie, murine-specific host range), and is replication-competent. Thus, only the first-stage adenovirus stage is capable of infecting human (HeLa) cells, whereas only the second-stage retrovirus produced from the initially infected cells is capable of replicating in murine (NIH3T3) cells as confirmed by spread of GFP fluorescence and Southern blot.[br]In our second-generation hybrid vectors, the first-stage helper-dependent adenovirus encodes a complete packaging system consisting of three separate components (an HIV- or MLV-based gag-pol expression cassette, an amphotropic env expression cassette, and a replication-defective lentivirus or retrovirus vector cassette carrying a lacZ marker and HSV thymidine kinase suicide gene). With these vectors, we have been able to demonstrate the two-stage mechanism by use of a GFP transgene unlinked to the retro/lentivirus components as a specific marker for the first-stage adenovirus, which have currently been amplified to titers on the order of 10e7-8 GFP transducing units per ml, and subsequent transmission of the lacZ marker alone indicating second-stage retro/lentivirus-mediated gene transfer. The retro/lentivirus second stage vector can be produced at titers on the order of 10e5 lacZ transducing units per ml after transduction of 10e6 target cells by a single copy of the first stage adenovirus at MOI=1. Thus, the first stage adenovirus transiently converts the initially transduced target cells to retro/lentivirus producer cells, and thereby achieves significant amplification of the initial input titer through in situ production of the second stage vector. This titer amplification effect may also prove useful in overcoming the production difficulties encountered in applications of helper-dependent adenovirus vectors, and by achieving permanent transduction of the adjacent target cells through the second stage retro/lentivirus, this hybrid vector system has the potential utility for gene transfer applications in which long term transgene expression is required. |
