TY - BOOK

T1 - Optimization of retroviral packaging cells for scale-up of vector production

AU - Heuvel, Yasemin van

PY - 2023

Y1 - 2023

N2 - Retroviral vectors (RVVs) are the key players for somatic gene therapy. However, the large-scale production is very time- and cost-intensive due to the production in adherent cell lines and transient plasmid transfections. To facilitate large-scale RVV production, the bioprocess engineering procedures need to be optimized. In this thesis, ecotropic murine leukemia virus (MLV)-based viral packaging cells (VPCs) were generated and procedures for the rapid and stable production of viral vectors in suspension were developed and improved. For this purpose and as a proof-of-concept (POC), suspension human embryonic kidney cells growing in serum-free media, were employed. In addition, a hybrid technology based on the Sleeping Beauty (SB) vector system and MLV donor vector components was applied for an efficient and rapid cell transfection. After stringent selection, a stable polyclonal VPC was generated within only three weeks. The resulting cells were able to produce the ecotropic MLV-based vectors at high levels and for months. The viral functional titers measured in murine fibroblasts and murine myeloblasts exceeded previous published titers generated with adherent or suspension VPCs, reaching tenfold higher titers of up to 1.4 × 10^7 transducing units per mL (TU/mL). Efficient gene transfer was also demonstrated in pre-clinically relevant murine hematopoietic stem and progenitor cells (HSPC). To further increase the production volume, as well as to advance the transposition technology, the transposase gene was in vitro synthesized to mRNA and co-transfected with the respective transposon-MLV vector components. Thus, no relevant genes for MLV vector production could be remobilized from the VPC genome any longer by a possibly genetically resident transposase, securing VPC stability. The stable suspension VPCs also proved to be highly efficient and viral titers were improved tenfold as compared to simple plasmid-based transfection or plasmid-based transposase transposition, exceeding 5 × 10^7 TU/mL. Finally, the VPC was successfully cultivated in a stirred-tank bioreactor (STR) with a 500 mL culture volume, continuously producing functional vectors during a ten-day process. This technology will enable future generations of stable VPCs and clinically relevant viral vector productions in fully automated STRs.

AB - Retroviral vectors (RVVs) are the key players for somatic gene therapy. However, the large-scale production is very time- and cost-intensive due to the production in adherent cell lines and transient plasmid transfections. To facilitate large-scale RVV production, the bioprocess engineering procedures need to be optimized. In this thesis, ecotropic murine leukemia virus (MLV)-based viral packaging cells (VPCs) were generated and procedures for the rapid and stable production of viral vectors in suspension were developed and improved. For this purpose and as a proof-of-concept (POC), suspension human embryonic kidney cells growing in serum-free media, were employed. In addition, a hybrid technology based on the Sleeping Beauty (SB) vector system and MLV donor vector components was applied for an efficient and rapid cell transfection. After stringent selection, a stable polyclonal VPC was generated within only three weeks. The resulting cells were able to produce the ecotropic MLV-based vectors at high levels and for months. The viral functional titers measured in murine fibroblasts and murine myeloblasts exceeded previous published titers generated with adherent or suspension VPCs, reaching tenfold higher titers of up to 1.4 × 10^7 transducing units per mL (TU/mL). Efficient gene transfer was also demonstrated in pre-clinically relevant murine hematopoietic stem and progenitor cells (HSPC). To further increase the production volume, as well as to advance the transposition technology, the transposase gene was in vitro synthesized to mRNA and co-transfected with the respective transposon-MLV vector components. Thus, no relevant genes for MLV vector production could be remobilized from the VPC genome any longer by a possibly genetically resident transposase, securing VPC stability. The stable suspension VPCs also proved to be highly efficient and viral titers were improved tenfold as compared to simple plasmid-based transfection or plasmid-based transposase transposition, exceeding 5 × 10^7 TU/mL. Finally, the VPC was successfully cultivated in a stirred-tank bioreactor (STR) with a 500 mL culture volume, continuously producing functional vectors during a ten-day process. This technology will enable future generations of stable VPCs and clinically relevant viral vector productions in fully automated STRs.

U2 - 10.15488/14856

DO - 10.15488/14856

M3 - Doctoral thesis

CY - Hannover

ER -