Background: Human gene therapy vectors based on primate lentiviruses harbour in contrast to oncoretroviruses the risk of vector mobilization by human immunodeficiency viruses (HIV). Infection of cells transduced with a lentiviral vector by HIV could lead to packaging of the lentiviral vector RNA into HIV particles and transfer of the vector.
Methods: A new approach based on primer complementation was developed to reduce the risk of vector mobilization. The primer binding site (PBS) of an SIV-based vector was mutated abolishing tRNA primer binding and thus blocking reverse transcription. This block was efficiently by-passed during vector production by providing an artificial tRNA matching the mutated PBS with titers reaching 10(6) infectious units/ml.
Results: Primer-complemented SIV vectors were mobilized from transduced cells by HIV-1 >150-fold less efficiently than vectors with wild-type PBS. Mobilization of the primer-complemented SIV vector by SIV was inhibited to a lesser extent indicating reduced efficacy of the primer complementation approach for preventing mobilization of lentiviral vectors by homologous virus. The analysis of the PBS of the vector DNA in target cells transduced with vectors containing mutated PBS in the absence of a matched tRNA suggests that formation of heterozygous particles followed by priming on the helper RNA and strand switch during reverse transcription can lead to mobilization of the primer-complemented vector by SIV, but not HIV-1. Although self-inactivating vectors were more efficient in preventing vector mobilization by HIV-1 than primer-complemented vectors, mobilization remained undectable only if both approaches were combined.
Conclusions: The primer complementation approach should further reduce the risk of mobilization of self-inactivating SIV-based vectors by HIV-1 and thus increase their safety.
Copyright 2004 John Wiley & Sons, Ltd.