A replication competent lentivirus (RCL) assay for equine infectious anaemia virus (EIAV)-based lentiviral vectors

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.

Development of inducible EIAV-based lentiviral vector packaging and producer cell lines

“…K2800-20). This plasmid was digested with BglII/PciI and the bsr gene isolated and inserted into pONYK1 39 that had been digested with BspHI/PciI thereby replacing the kanamycin resistance gene with the bsr gene.…”

Development of an Equine-Tropic Replication-Competent Lentivirus Assay for Equine Infectious Anemia Virus-Based Lentiviral Vectors

“…No human cell line is able to support replication of EIAV because of the lack of receptor(s) (e.g., equine lentivirus receptor-1 [ELR1]; Zhang et al, 2005) and the low activity of the LTR enhancer/promoter in human cells because of the inability of equine Tat to recruit human cyclin T1 to the transcription complex (Bieniasz et al, 1999). Our efforts in developing an EIAV-permissive HEK293-based cell line coexpressing equine cyclin-T1 (eCT1) (Miskin et al, 2006) and equine lentivirus receptor-1 (ELR1) (our unpublished data) have been met with major challenges that, although interesting, are beyond the scope of this paper to describe.…”

“…This testing approach takes into consideration the potential for unpredictable recombination of vector components with other nucleic acid sequences within the production cells, such as endogenous retroviruses and/or therapeutic gene sequences. The nonpermissive nature of human cell cultures to EIAV precludes the development of a human cell-based RCL assay coupled with an EIAV-derived positive control, and consequently we previously validated an assay using amphotropic murine leukemia virus (MLV) as a surrogate positive control and HEK293 cells as amplification cells (Miskin et al, 2006). However, after consultation with the Food and Drug Administration (FDA), and with a desire to comprehensively demonstrate the safety of the EIAV lentiviral vector system, we now report the development of an additional Good Manufacturing Practice (GMP)-compliant RCL assay based on equine cells and using an EIAV-derived positive control virus.…”

“…In addition, we generated attenuated EIAV mutants, each functionally lacking one of the three accessory genes, and tested their replication in an attempt to model putative RCLs that might arise from the minimal vector system. The equine-tropic RCL assay (ET-RCL assay) described herein follows a format similar to that described for the MLV/HEK293 RCL assay: cell culture phase amplification of putative RCL within final drug product (FDP) or EOPCs, followed by an end-point analysis of reverse transcriptase activity in supernatants by quantitative RT-PCR (Miskin et al, 2006).…”

“…The sensitivity of RCL detection must be enhanced by an amplification phase, requiring a permissive cell type, followed by a sensitive indicator or end-point assay (Escarpe et al, 2003;Sastry et al, 2003Sastry et al, , 2005Segall et al, 2003); for a review see Sastry and Cornetta (2009). The highly sensitive product-enhanced reverse transcriptase (PERT) assay (a qRT-PCR assay) is used for the relative titration of vector lots and as an end-point assay for RCL detection (Martin-Rendon et al, 2002;Miskin et al, 2006). Testing for RT activity is considered to be the most sensitive and appropriate readout because, whatever its genomic configuration, by definition any RCL must possess RT activity.…”