Viral vectors are a common tool for introducing new or corrected genes into human cells tor product cellular therapy products. Chimeric antigen receptor T (CAR T) cells require lentivirus to introduce the CAR gene into activated T cells. Lentiviral vectors have also been used for the treatment of hereditary disorders, such as severe combined immunodeficiency disorder (SCID) and Wiskott-Aldrich syndrome. Although lentiviral vectors are ideal for delivery and stable integration of genes of interest into the host cell genome, they potentially pose risks to human health, such as integration-mediated transformation and generation of a replication-competent lentivirus (RCL) capable of infecting non-target cells. In consideration of the latter risk, all cell-based products modified by lentiviral vectors and intended for patient use must be tested for RCL before treatment of the patient.

Lentivirus vectors can be used/made following Biosafety Level 2 (BSL2) containment criteria as follows:

• The vector is minimal containing LTRs, packaging sequencing, and no coding gene sequences;
• A heterologous envelope is used (not HIV or SIV);
• The packaging plasmid has no sequences on the LTR that could recombine;
• Genes inserted do not code for oncogenes, oncogene precursor, or highly toxic molecules.

QVirus™ Platform at Creative Biogene provides reliable and affordable RCL test for lentiviral vector stocks. With many years of experience in providing fully integrated biosafety testing for these programs, we have supported a great number of successful regulatory submissions.

Assay method for detection of Replication Competent Lentivirus

• Cell-based assay

The current standard for RCL testing is a cell-based assay by using a permissive cell line to allow for expansion of low-level virus followed by detection of viral proteins. RCL amplification is performed by culturing vector containing supernatant or vector-producing cells on a permissive cell line. The cultures are maintained over various passages in order to amplify any low level infectious virus and also to remove vector associated Reverse Transcriptase (RT) activity by dilution. The cell culture supernatant fluids from the later passages are harvested and the final passage supernatant is typically assayed for the presence of retrovirus through Product Enhanced Reverse Transcriptase (PERT) assay.

• qPCR-based assays

QVirus™ Platform has developed the qPCR-based assays for quick determination of RCL presence in order to have fresh infusion of the cell product. With the increasing reliance on rapid (PCR) testing for RCL, it is significant to develop assays that are reproducible, accurate, and have robust sensitivity while maintaining a low rate of false positives in order to ensure adherence to patient safety standards.

• Lentiviral vector stocks assays

Lentiviral vector stocks are tested for the presence of RCL through monitoring p24 antigen expression in the culture medium of transduced 293T cells. Serial passaging of the transduced p24 cells over this period allows for the amplification of RCL. This method is used to assess the presence of RCR in lentiviral preparations.