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Replication-Competent Lentivirus Testing

Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient in vitro and in vivo gene delivery and long-term transgene expression in both dividing and nondividing cells. They have the advantages of a large cloning capacity, the lack of a requirement for viral gene expression, and the capacity to integrate into the target cell genome for stable transduction. In addition, they are highly efficient for in vivo gene delivery in a broad range of hosts cells, such as neurons, endothelial cells, hepatocytes, and hematopoietic stem cells.

Despite the strong rationale for their utilization in human gene therapy and extensive preclinical data, concerns regarding their safety have hampered their testing in clinical trials. A sensitive assay for the detection of replication-competent lentiviruses (RCL) in large-scale preparations of HIV-based lentiviral vectors is needed.

The major safety concerns regarding lentivirus vector manufacture and clinical use are listed below:

  1. Potential generation of replication competent lentivirus (RCL) during the production process.
  2. In vivo recombination with endogenous lentiviral sequences.
  3. Insertional mutagenesis of proviral DNA in, or close to, active genes which may trigger tumor initiation or promotion.

Creative Biogene, as a leader in viral service, provides reliable and affordable replication-competent lentivirus (RCL) test for lentiviral vector stocks. With many years of experience in providing fully integrated biosafety testing for these programs, we have supported a vast number of successful regulatory submissions.

1. Standard reverse transcriptase assays

HIV type 1 (HIV-1) is a positive single-strand RNA virus and the genome encoded reverse transcriptase (RT) is essential for HIV-1 replication. Due to the essential role in the viral life cycle and clear functional and structural investigation, HIV-RT has been the target of considerable interest for lentivirus detection.

2. PERT assays

Product-enhanced reverse transcriptase (PERT) assays were developed to increase the sensitivity. The PERT assay involves producing cDNA from template RNA by suspected RT in a test sample, amplifying the cDNA by PCR and detecting the PCR products by gel electrophoresis or Southern blotting. FDA has requested the use of PERT assay to detect viral activity in human viral vaccine products.

3. Lentiviral vector stocks assays

Lentiviral vector stocks are tested for the presence of replication-competent lentivirus (RCL) by 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 assay is used to assess the presence of RCR in lentiviral preparations.


  1. R&D studies
  2. GLP/GMP submissions
  3. Commercially Manufactured CAR T cells
  4. Lentiviral vector-based gene therapy programs


  1. Hizi A, Tal R, Shaharabany M, et al. Specific inhibition of the reverse transcriptase of human immunodeficiency virus type 1 and the chimeric enzymes of human immunodeficiency virus type 1 and type 2 by nonnucleoside inhibitors[J]. Antimicrobial agents and chemotherapy, 1993, 37(5): 1037-1042.
  2. US FDA:

  3. Guidance for Industry: Guidance for Human Somatic Cell Therapy and Gene Therapy. 1998.
  4. Guidance for Industry: Supplemental guidance on testing for replication competent retrovirus in retroviral vector based gene therapy products and during follow-up of patients. 2006.
  5. Guidance for FDA reviewers and sponsors. Content of and review of chemistry, manufacturing and control information for human gene therapy IND applications. 2008.
  6. EU:

  7. The European Medicines Agency (EMA) Evaluation of medicines for Human Use. Committee for Medicinal Products for Human Use (CHMP). Guideline on development and manufacture of lentiviral vectors. EMEA/CPMP/BWP/2458/03. 2005.
  8. Quality, preclinical and clinical aspects of gene transfer medicinal products. CHMP/GTWP/234523/09. 2009.

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