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.

The product-enhanced reverse transcriptase (PERT) assay has been used to detect reverse transcriptase (RT) activity associated with retroviruses. Although the PERT assay has been proposed as a method for detection of replication-competent retrovirus (RCR) and lentivirus (RCL), it has not been rigorously compared with existing methods for RCR and RCL detection. We have assessed the PERT assay for detection of RCL and RCR that may contaminate lentiviral and retroviral vectors and compared it with published methods for RCL (p24(gag) ELISA/(gag) PCR) and RCR (S+/L-) detection. Our results suggest that the PERT assay is as sensitive as p24gag ELISA and gag PCR for detection of replication-competent HIV-1 in an RCL detection assay. Comparison of detection of replication-competent retroviruses, GALV and RD114, by extended S+/L- and PERT assays indicates that both assays can detect 1 IU of each virus. Our findings suggest that the PERT assay can be used for RCL and RCR testing of a variety of retroviral vectors regardless of the structure, sequence, and envelope of the vectors.

The ability of lentiviral-based vectors to integrate into non-dividing cells such as neuronal cells has been exploited to correct a number of disease models. Using vectors based on the equine infectious anaemia virus (EIAV) we have previously reported the correction of an animal model of Parkinson's disease (Azzouz et al. J Neurosci. 2002 Dec 1;22(23):10302–12). To use such vectors in the clinic each manufactured lot must be tested and demonstrated to be free of replication competent lentivirus (RCL). We have designed the vectors in such a way to limit the possibility of homologous recombination taking place; the vector genome contains only 824 nt of EIAV sequence with no coding regions; all the accessory genes have been removed entirely from the system; the gag/pol sequence has been codon optimised and a heterologous envelope is used (VSV-G). However, there is still the formal possibility of some form of recombination leading to the generation of an RCL. We have therefore developed an RCL assay based on the property common to all members of the Retroviradae family, namely the presence of reverse transcriptase (RT). RT activity can be detected by a sensitive assay: Product Enhanced Reverse Transcriptase (PERT). The test article is passaged on human cells to amplify any replicating entity and then a PERT assay is carried out on the supernatant. A typical set of assay results will be presented.