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Cellular contaminants of adeno-associated virus vector stocks can enhance transduction
L Tenenbaum1, M Hamdane, M Pouzet
1IRIBHN, Campus Erasme, Université Libre de Bruxelles, Germany.
Abstract:
Transduction efficiency of different types of recombinant (r)AAV-2 based vectors preparations markedly differed, with apparently no correlation with the replicative titers. Using HeLa cells as target for transduction, 105 and 30 infectious units were necessary to observe one transductant using respectively cesium-chloride-purified rAAV and crude lysates of producer cells obtained by sonication. The purified vectors were however able to transduce HEK-193 cells efficiently, but transgene expression was detected with some delay compared with crude lysates. The unexpected high transduction efficiency of sonicated crude lysates was due to virally mediated gene transfer, since similar sonicated crude lysates, but with no AAV rep and cap genes, did not lead to detection of transgene products after incubation with HeLa cells. Furthermore, sonicated cellular extracts of 293 or 293/T cells given in trans stimulate transduction of HeLa cells by purified rAAV. In contrast, neither extracts from the adenovirus E1-transformed 911 cell line, nor from other cell lines not harboring any adenovirus gene, had enhancing effect on rAAV-mediated transduction. These data suggest that 293 sonicated extracts contain factors which stimulate rAAV-mediated transduction of cells that are normally poorly transduced and offer a system to identify such factors and to characterize further the steps limiting the transfer of gene by AAV vectors.
Insights
Recombinant adeno-associated virus (rAAV) vector preparations show variable transduction efficiency. Sonicated cell extracts from specific cell lines enhance rAAV transduction in poorly transduced cells.
Area of Science:
- Molecular Biology
- Gene Therapy
- Virology
Background:
- Adeno-associated virus (AAV) vectors are promising tools for gene therapy.
- Transduction efficiency of recombinant AAV (rAAV) vectors can vary significantly.
- Understanding factors influencing rAAV transduction is crucial for optimizing gene delivery.
Purpose of the Study:
- To investigate the reasons behind the variable transduction efficiency of different rAAV-2 vector preparations.
- To identify cellular factors that may enhance rAAV-mediated gene transfer.
- To characterize the limiting steps in AAV vector gene transfer.
Main Methods:
- Comparison of transduction efficiency using cesium-chloride-purified rAAV and crude cell lysates.
- Transduction assays using HeLa and HEK-193 cells.
- Analysis of transgene expression following transduction with various rAAV preparations and cellular extracts.
- In trans complementation assays using sonicated cellular extracts from different cell lines.
Main Results:
- Transduction efficiency of rAAV preparations did not correlate with replicative titers.
- Crude lysates showed higher initial transduction efficiency in HeLa cells compared to purified rAAV.
- Sonicated cellular extracts from 293 or 293/T cells significantly enhanced rAAV transduction of HeLa cells.
- Extracts from cell lines lacking adenovirus genes did not enhance transduction.
Conclusions:
- Cellular factors present in 293/T cell extracts enhance rAAV transduction, particularly in cells that are typically poorly transduced.
- These findings suggest a system for identifying factors that improve AAV vector gene delivery.
- Further characterization of these factors can elucidate critical steps in AAV-mediated gene transfer.