An Improved Helper Plasmid Containing Deletions Within the E4 and E2a Genes Results in Increased Adeno-Associated Virus Productivity.
Hum Gene Ther
; 35(17-18): 767-776, 2024 Sep.
Article
en En
| MEDLINE
| ID: mdl-39001832
ABSTRACT
The use of a helper plasmid to replace adenovirus infection for adeno-associated virus (AAV) manufacturing has been common practice for decades. Adenovirus E4, E2a, and VA RNA genes are sufficient to support efficient AAV replication. In an effort to ensure that all transfected DNA has a functional role in AAV production, deletions were introduced to the E4 and E2a genes to determine if any portions were dispensable. Although a 900 bp deletion in the E2a intron did not have an impact, the removal of open reading frames (orf) 1-4 from the E4 gene resulted in a doubling of AAV productivity. The E4Δorf1-4 deletion was associated with a reduction in E4orf6 transcripts, along with an increase in Rep and Cap transcripts and protein levels, which corresponded to increased AAV productivity in crude lysate. The final product of these studies was a helper plasmid, termed OXB-Helper_3, that is >3.4 kb smaller than the original control plasmid and resulted in â¼2× improvement in vector genome productivity across multiple capsid serotypes, genome designs, and transfection platforms.
Palabras clave
Texto completo:
1
Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Plásmidos
/
Dependovirus
/
Vectores Genéticos
Límite:
Humans
Idioma:
En
Revista:
Hum Gene Ther
Asunto de la revista:
GENETICA MEDICA
/
TERAPEUTICA
Año:
2024
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos