Gene therapy products testing 
Gene therapy first appeared in 1990 and is now almost ready for large-scale therapeutic use. The proportion of clinical trials using viral vectors or genetically modified cells has steadily grown over the last decade. This progress has been accompanied by regulatory recommendations concerning viral biosafety.
To assist with the biosafety testing of gene therapy products, Texcell has developed a wide range of tests for detecting replication-competent viruses and contaminating viruses.
Assays for Replication competent retroviruses (RCR)
As indicated in the FDA note “Supplemental Guidance on Testing for Replication-competent Retrovirus in Retroviral Vector-Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors”, the quantity of material that should be tested for RCR depends on the nature of the test product.
Five per cent of total supernatants may be tested by amplification in a permissive cell line. In cases where the supernatant volume exceeds 6 litres, an alternative approach may be applied: sufficient supernatant must be tested to ensure a 95% probability of detecting RCRs if present at a concentration of 1 RCR/100 ml. At this concentration, there is a 95% probability that about 300 ml will contain an RCR. Therefore, assuming the assay is sensitive enough to detect a single RCR, 300 ml is sufficient.
Texcell has developed very sensitive RCR assays that comply with these recommendations. These assays include an internal positive control that gives equivalent validated results with the VA1450 reference strain (ATCC).
For cells, 1% of total pooled cells or 108 cells (whichever is smaller) should be co-cultured with a permissive cell line.
For all the protocols described below involving the culture of supernatant or co-culture of cells with a permissive cell line, a minimum of five passages is carried out to amplify any potential RCR present.
Assays for murine RCR
Protocol 1170-S: Detection of murine RCRs in vector stock
RCR is detected by amplifying the vector stock in Mus dunni cells for five passages, followed by the PG4 S+L- assay.
RCR is detected by co-cultivating the vector-producer cells with Mus dunni cells for five passages, followed by the PG4 S+L- assay.
Protocol 1178-S: Detection of murine RCR in patient samples using a mobilisation assay
The mobilisation assay detects murine RCR by inoculating Mus dunni Bag cells with the test sample, followed by five passages. The supernatant is then inoculated on Mus dunni for RCR development.
Protocol 1178-C: Detection of murine RCR in patient cells using a mobilisation assay
The mobilisation assay detects murine RCR by co-cultivating the patient cells with Mus dunni Bag cells, followed by five passages. The supernatant is then inoculated on Mus dunni for RCR development.
Assay for GaLV RCR
Protocol 1188-S: Supra/extended assay for detection of GaLV RCR in vector stocks
RCR is detected by amplifying the vector stock in HEK293 for two or five passages respectively, followed by the PG4 S+L- assay.
RCR is detected by co-cultivating the vector-producer cells with HEK293 for two or five passages respectively, followed by the PG4 S+L- assay.
Protocol 1183-S: Detection of GaLV RCR in vector stocks using a mobilisation assay
The mobilisation assay detects GaLV RCR by inoculating HEK 293 Bag with vector stock, followed by five passages and development on HEK 293.
Protocol 1183-C: Detection of GaLV RCR in vector-producer cells using a mobilisation assay
The mobilisation assay detects GaLV RCR by co-cultivating the vector-producer cells with HEK 293, followed by five passages. The supernatant is then inoculated on HEK 293 Bag, with RCR development performed on HEK 293.
Assay for replication competent adenoviruses (RCA)
To test for the presence of replication-competent adenoviruses (RCAs), even at very low concentrations, we have developed a screening method based on RCA amplification. This rapid and sensitive method has no adverse effects on high-titre vectors. To comply fully with the recommendations published by the FDA in its note “Guidance for Human Somatic Cell Therapy and Gene Therapy”, any wild-type adeno-associated virus (AAV) contaminating the master cell bank, the master virus seed stock and the final product is detected in our laboratory using PCR-based techniques.
The regulatory bodies generally state that there should be less than 1 RCA per 1x108 PFU of vector. To respond to this request, Texcell has developed a range of assays for RCA detection.
Protocol 1300: Direct RCA assay
A549 cells are used to detect RCAs in the vector stock. At least three weeks after inoculation of A549 with the vector stock, the presence/absence of plaque-forming units (PFU) is noted. This method can detect 1 RCA per 1x108 PFU of vector.
Protocol 1301: Extended RCA assay 109
RCAs are detected after amplification of the vector stock in A549 cells, followed by an A549 assay. This method can detect 1 RCA per 1x109 PFU of vector.
Protocol 1302: Extended RCA assay 1010
RCAs are detected after amplification of the vector stock in A549 cells, followed by an A549 assay. This method can detect 1 RCA per 1x1010 PFU of vector.
Protocol 1064: PCR-based Detection of Adeno-Associated Viruses (DNA)
Protocol 1071: PCR–based Detection of Adenoviruses (DNA)
Protocol 1080: RTQ-PCR-base Detection of Adenovirus type 5 (DNA)
Assay for replication competent lentivoruses (RCL)
Texcell has developed customised protocols for detecting RCLs.
Do not hesitate to contact us for further information on these assays.
