Cell Engineering Strategies in HEK293 cells to enhance rAAV production and secretion
Ruiz Ayala, Carlos
Cervera Gracia, Laura, tut. (Universitat Autònoma de Barcelona)
Universitat Autònoma de Barcelona. Escola d'Enginyeria

Data:	2025
Resum:	Recombinant adeno-associated virus (rAAV) vectors have emerged as a leading platform for in vivo gene therapies due to their favorable safety profile, broad tissue tropism, and long-lasting transgene expression. However, the high production cost of rAAV remains a major barrier to clinical scalability and patient accessibility. Transient transfection (TT) in HEK293 cells is currently the most widely used method for rAAV manufacturing, but it presents limitations in terms of scalability, batch-to-batch variability, and overall cost. In this context, the Extended Gene Expression (EGE) protocol has demonstrated improved production yields by prolonging the expression window. Notably, an increase in rAAV titers was observed in the supernatant fraction, suggesting a greater extracellular release of viral particles. This may reduce the need for cell lysis during rAAV production, simplifying downstream processing and enhancing process efficiency. This work explores a metabolic engineering approach to further enhance rAAV secretion by targeting host cell factors involved in extracellular vesicle (EV) biogenesis. In previous proteomic studies comparing TT and EGE conditions, a upregulation of three genes, SNAP47, SMPD3, and VPS37B, was observed in the EGE condition and they were selected for cell engineering due to their involvement in membrane trafficking, vesicle formation, and EV release pathways. A HEK293 stable cell pool was generated via random integration of SNAP47 gene using antibiotic selection. Transient transfection and EGE protocols were then performed to evaluate rAAV9 production. The results showed that overexpression of SNAP47 enhanced rAAV9 yield in the supernatant fraction, suggesting improved extracellular release. These findings support the role of vesicle-related pathways in rAAV secretion and demonstrate that targeted metabolic engineering can increase production efficiency. Overall, this study lays the groundwork for developing stable HEK293 cell lines with enhanced rAAV secretion capacity. This strategy offers a promising route to more cost-effective and scalable rAAV manufacturing platforms, which could ultimately improve the availability of gene therapies for broader patient populations.
Drets:	Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, i la comunicació pública de l'obra, sempre que no sigui amb finalitats comercials, i sempre que es reconegui l'autoria de l'obra original. No es permet la creació d'obres derivades.
Llengua:	Anglès
Titulació:	Bioquímica, Biologia Molecular i Biomedicina [4313794]
Pla d'estudis:	Màster Universitari en Bioquímica, Biologia Molecular i Biomedicina [1599]
Document:	Treball de fi de postgrau
Matèria:	Fabricació de teràpies gèniques ; Producció de rAAV ; Enginyeria celular de cèl·lules HEK293 ; Enginyeria metabòlica ; Desenvolupament de línies celulars estables ; Fabricaciónde terapias génicas ; Producción de rAAV ; Ingeniería celular de células HEK293 ; Ingenería metabòlica ; Desarrollo de líneas celulares estables ; Gene therapy manufacturing ; Raav production ; HEK293 cell engineering ; Metabolic engineering ; Stable cell line development

31 p, 1.4 MB

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