Product Enhanced Reverse Transcriptase for assessing replication competent virus in vectors retroviral vectors pseudotyped with GALV and VSV-G envelopes.
J Virol Methods
; 329: 114988, 2024 Sep.
Article
en En
| MEDLINE
| ID: mdl-38908550
ABSTRACT
We evaluated the use of the Product Enhanced Reverse Transcriptase (PERT) assay as a means of detecting virus in retroviral vectors products pseudotyped with Gibbon Ape Leukemia Virus (GALV) and Vesicular Stomatitis Virus G (VSVG) envelopes. PERT provides greater standardization than the S+/L- assay which has been used extensively in virus detection. A challenge is that PERT will also detect residual retroviral vectors as vector particles contain reverse transcriptase. Vector products were cultured for 3 weeks on HEK293 cells to amplify any potential virus. In addition, vector supernatant and end-of-production cells were spiked with GALV to evaluate for inhibition by the test article. Results of PERT and the S+/L- assay were compared. PERT and S+/L- assays were both effective in detecting virus. Vector supernatants were negative at the end of 3 weeks of culture by PERT for both GAVL and VSVG pseudotyped vector. In contrast, end-of-production cells were positive by PERT due to persistent vector producing cells. A one-week culture of cell-free media obtained at the 3 weeks timepoint allowed distinction of virus-free test articles from those with virus. The PERT assay is suitable for detecting replication competent retrovirus in vector products pseudotyped with GALV and VSVG envelopes.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
ADN Polimerasa Dirigida por ARN
/
Virus de la Leucemia del Gibón
/
Vectores Genéticos
Límite:
Humans
Idioma:
En
Revista:
J Virol Methods
Año:
2024
Tipo del documento:
Article
Pais de publicación:
Países Bajos