Development of a replication-competent lentivirus assay for dendritic cell-targeting lentiviral vectors

Abstract

It is a current regulatory requirement to demonstrate absence of detectable replication-competent lentivirus (RCL) in lentiviral vector products prior to use in clinical trials. Immune Design previously described an HIV-1-based integration-deficient lentiviral vector for use in cancer immunotherapy (VP02). VP02 is enveloped with E1001, a modified Sindbis virus glycoprotein which targets dendritic cell-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) expressed on dendritic cells in vivo. Vector enveloped with E1001 does not transduce T-cell lines used in standard HIV-1-based RCL assays, making current RCL testing formats unsuitable for testing VP02. We therefore developed a novel assay to test for RCL in clinical lots of VP02. This assay, which utilizes a murine leukemia positive control virus and a 293F cell line expressing the E1001 receptor DC-SIGN, meets a series of evaluation criteria defined in collaboration with US regulatory authorities and demonstrates the ability of the assay format to amplify and detect a hypothetical RCL derived from VP02 vector components. This assay was qualified and used to test six independent GMP production lots of VP02, in which no RCL was detected. We propose that the evaluation criteria used to rationally design this novel method should be considered when developing an RCL assay for any lentiviral vector.

Detection of Replication Competent Lentivirus Using a qPCR Assay for VSV-G

“…RCL of third-generation lentiviral vectors has never been observed,18, 20, 21 but the most likely scenario for formation of RCL would be through recombination of the transfer plasmid with the supporting packaging plasmids 22 8, 10, 13, 23.…”

Design of a titering assay for lentiviral vectors utilizing direct extraction of DNA from transduced cells in microtiter plates

“…Previous work has demonstrated that this cell line is transduced by VP02 in a DC-SIGN-dependent manner. 3 , 11 To maximize sensitivity of the spin column method, a wide range of cell inputs within the limits suggested by the manufacturer was evaluated (1.56 × 10 4 – 1.00 × 10 6 cells/column), each of which was eluted in a final fixed volume of 400 µl, as suggested by the manufacturer to maximize DNA yield. In comparison, in the in situ extraction method, the starting cell number is fixed.…”

Absence of Replication-Competent Lentivirus in the Clinic: Analysis of Infused T Cell Products

“…33,34 To date, the most common assays for screening gene therapy products are assays that combine an amplification phase, using a cell line capable of expanding attenuated viruses to high titer, with subsequent detection of virus using ELISA or molecular assays. 29,[35][36][37][38][39] Since RCLs arising during vector production are still theoretical, their growth rate is unknown, but it is likely to be significantly attenuated, compared to wild-type lentiviruses, due to the absence of accessory genes. 33 Therefore, regulators have required biologic assays to utilize an extended culture period of approximately 3 weeks (a minimum of 5 passages) 27 to amplify any slow-growing viruses.…”