Construction of stable producer cells to make high-titer lentiviral vectors for dendritic cell-based vaccination.
Biotechnol Bioeng
; 109(6): 1551-60, 2012 Jun.
Article
en En
| MEDLINE
| ID: mdl-22179950
ABSTRACT
Lentiviral vectors (LVs) enveloped with an engineered Sindbis virus glycoprotein can specifically bind to dendritic cells (DCs) through the surface receptor DC-SIGN and induce antigen expression, thus providing an efficient method for delivering DC-directed vaccines. In this study, we constructed a stable producer line (LV-MGFP) for synthesizing DC-SIGN-targeted HIV-1-based LVs (DC-LVs) encoding green fluorescent protein (GFP) by a concatemeric array transfection technique. We demonstrated that the established stable clones could routinely produce vector supernatants with titers above 10(7) transduction units per milliliter (TU/mL) during a continuous 3-month cell passage. The producer cells were also capable of generating similar titers of DC-LVs in serum-free medium. Moreover, the addition of 1-deoxymannojirimycin (DMJ) enabled the producer cells to manufacture DC-LVs with both improved titers and enhanced potency to evoke antigen-specific CD8(+) T cell responses in mice. The stable lines could accommodate the replacement of the internal murine stem cell virus (MSCV) promoter with the human ubiquitin-C (Ubi) promoter in the lentiviral backbone. The resulting DC-LVs bearing Ubi exhibited the enhanced potency to elicit vaccine-specific immunity. Based on accumulated evidence, our studies support the application of this production method in manufacturing DC-LVs for preclinical and clinical testing of novel DC-based immunization.
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Transducción Genética
/
Biotecnología
/
Células Dendríticas
/
VIH-1
/
Tecnología Farmacéutica
/
Vacunación
/
Vectores Genéticos
Límite:
Animals
Idioma:
En
Revista:
Biotechnol Bioeng
Año:
2012
Tipo del documento:
Article
País de afiliación:
Estados Unidos