Enhanced ER protein processing gene expression increases rAAV yield and full capsid ratio in HEK293 cells.
Appl Microbiol Biotechnol
; 108(1): 459, 2024 Sep 04.
Article
in En
| MEDLINE
| ID: mdl-39230729
ABSTRACT
The recombinant adeno-associated virus (rAAV) vector is among the most promising viral vectors in gene therapy. However, the limited manufacturing capacity in human embryonic kidney (HEK) cells is a barrier to rAAV commercialization. We investigated the impact of endoplasmic reticulum (ER) protein processing and apoptotic genes on transient rAAV production in HEK293 cells. We selected four candidate genes based on prior transcriptomic studies XBP1, GADD34 / PPP1R15A, HSPA6, and BCL2. These genes were stably integrated into HEK293 host cells. Traditional triple-plasmid transient transfection was used to assess the vector production capability and the quality of both the overexpressed stable pools and the parental cells. We show that the overexpression of XBP1, HSPA6, and GADD34 increases rAAV productivity by up to 100% and increases specific rAAV productivity by up to 78% in HEK293T cells. Additionally, more prominent improvement associated with ER protein processing gene overexpression was observed when parental cell productivity was high, but no substantial variation was detected under low-producing conditions. We also confirmed genome titer improvement across different serotypes (AAV2 and AAV8) and different cell lines (HEK293T and HEK293); however, the extent of improvement may vary. This study unveiled the importance of ER protein processing pathways in viral particle synthesis, capsid assembly, and vector production. KEY POINTS ⢠Upregulation of endoplasmic reticulum (ER) protein processing (XBP1, HSPA6, and GADD34) leads to a maximum 100% increase in rAAV productivity and a maximum 78% boost in specific rAAV productivity in HEK293T cells ⢠The enhancement in productivity can be validated across different HEK293 cell lines and can be used for the production of various AAV serotypes, although the extent of the enhancement might vary slightly ⢠The more pronounced improvements linked to overexpressing ER protein processing genes were observed when parental cell productivity was high, with minimal variation noted under low-producing conditions.
Key words
Full text:
1
Collection:
01-internacional
Database:
MEDLINE
Main subject:
Dependovirus
/
Endoplasmic Reticulum
/
X-Box Binding Protein 1
/
Genetic Vectors
Limits:
Humans
Language:
En
Journal:
Appl Microbiol Biotechnol
Year:
2024
Document type:
Article
Affiliation country:
Country of publication: