Lentiviral vectors have demonstrated great potential as gene therapy vectors mediating efficient in vitro and in vivo gene...
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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:
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.