1. ¹Oxford BioMedica (UK) Ltd, Medawar Centre, The Oxford Science Park, Oxford, UK
2. ²BioMedica Inc., San Diego, CA, USA

Correspondence: Dr J Miskin, Oxford BioMedica (UK) Ltd, Medawar Centre, The Oxford Science Park, Oxford OX4 4GA, UK. E-mail: j.miskin@oxfordbiomedica.co.uk

³Current address: Biotechnology Consulting Services, San Marcos, CA 92078, USA.

⁴Current address: Advantagene Inc., 382 Sunset Drive, Encinitas, CA 92024, USA.

Received 6 July 2005; Revised 9 September 2005; Accepted 12 September 2005; Published online 6 October 2005.

Abstract

Lentiviral vectors are being developed to satisfy a wide range of currently unmet medical needs. Vectors destined for clinical evaluation have been rendered multiply defective by deletion of all viral coding sequences and nonessential cis-acting sequences from the transfer genome. The viral envelope and accessory proteins are excluded from the production system. The vectors are produced from separate expression plasmids that are designed to minimize the potential for homologous recombination. These features ensure that the regeneration of the starting virus is impossible. It is a regulatory requirement to confirm the absence of any replication competent virus, so we describe here the development and validation of a replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based vectors. The assay is based on the guidelines developed for testing retroviral vectors, and uses the F-PERT (fluorescent-product enhanced reverse transcriptase) assay to test for the presence of a transmissible reverse transcriptase. We have empirically modelled the replication kinetics of an EIAV-like entity in human cells and devised an amplification protocol by comparison with a replication competent MLV. The RCL assay has been validated at the 20 litre manufacturing scale, during which no RCL was detected. The assay is theoretically applicable to any lentiviral vector and pseudotype combination.