Rapid Alternative to Standard Safety Tests Introduced

Early iterations of first-generation retroviral vectors have been associated with leukemia, but that risk has been brought to near zero with third-generation lentiviruses.

Scientists at Stanford University School of Medicine (Palo Alto, CA, USA) developed a rapid test to looks for a viral envelope marker in a sample of engineered cells that should not be there if there is no viral replication present. One advantage is that this can be done in the laboratory when the cultures are freshly prepared with some molecular reagents and a person working for a couple of hours. The standard procedure required by the US Food and Drug Administration (FDA, Silver Springs, MD, USA) waits 3 to 4 weeks to check if viral cultures grow, and samples need to be frozen and shipped to an outside testing site.

The investigators described the development and qualification of a molecular assay based on detection of envelope gene sequences (vesicular stomatitis virus G glycoprotein [VSV-G]) for replication competent lentivirus (RCL) in accordance with Minimum Information for Publication of Quantitative Real-Time polymerase chain reaction (qRT-PCR) Experiments (MIQE) guidelines. The results demonstrate the sensitivity, linearity, specificity, and reproducibility of detection of VSV-G sequences, with a low false-positive rate. These procedures are currently being used in our phase 1 clinical investigations. DNA was quantified on an Implen Nanophotometer, and A260/A280 and A260/A230 values were recorded as a representation of sample purity.

David L. DiGiusto, PhD, the senior author of the study, said, “A lot of people avoid this technology for rapid assay because it has this potential for false positives, but I think we've proven that it's very possible to have high sensitivity and high accuracy with this test. This method is pretty straightforward, and any lab using lentiviral vectors should consider running it on their lentiviral transduced cell products to document the lack of detectable replication-competent viruses.” The study was published on September 21, 2017, in the journal Molecular Therapy--Methods & Clinical Development.

Related Links:
Stanford University School of Medicine
US Food and Drug Administration