TY - BOOK

T1 - Development of transposon and retrovirus-derived vectors for the rapid establishment of efficient producer cell lines

AU - Tschorn, Natalie

PY - 2023

Y1 - 2023

N2 - With the growing demand for therapeutic proteins such as antibodies or vaccines, manufactures are in need to deliver products at high quality and constant productivity. For the biotechnological production of these products, mammalian cells are predominantly used. The productivity of producer cells is, amongst others, dependent on the number of integrated transgene copies per cell, and thus elevating stable gene transfer efficiencies to ensure sustained expression of the gene of interest is paramount. In this study the Sleeping Beauty transposon two-component vector system, consisting of a transposon donor plasmid and a transposase expression plasmid, was used to generate stable cell pools. However, using sensitive genomic PCR and reverse transcriptase PCR (RT-PCR), the transposase gene integration and expression was demonstrated for a time period of 48 days post transfection. To provide an alternative to the employment of plasmid-based transposase expression circumventing potential re-mobilization events of the already stably transposed transgenes, the transposase gene was transcribed in vitro into mRNA. After co-transfection of transposase transcripts at different ratios to the donor vector efficient transposition was obtained. This study demonstrated that this technical approach mediated high copy numbers and expression levels of transgenes in recombinant cells without the risk of undesired extended transposase expression. Besides transposons, retroviral vectors are frequently used to introduce foreign genetic material into mammalian cells aiming for the establishment of producer cells. Such viral vectors are commonly pseudotyped with VSV-G achieving high vector copy numbers in mammalian cells. However, this requires handling under BSL-2 conditions. To circumvent this, viral vectors were equipped with the ecotropic envelope protein PVC211mc, a molecular clone of Friend murine leukemia virus (MLV), enabling transduction of CHO cells and murine hematopoietic stem cells but allowing experiments in BSL- 1 laboratories. The aim of this work was to optimize gene transfer efficiencies by generating PVC211-derived envelope protein (Env) variants lacking the R-peptide and thus rendering the Env proteins fusogenic. All generated variants failed to efficiently pseudotype MLV vectors but two variants successfully pseudotyped HIV-1 particles. The HIV-1 vectors pseudotyped with the envelope variant eMLV-GaLVΔR mediated superior infectivity as compared to wild-type Env. HIV(eMLV-GaLVΔR) should prove useful as a tool for the establishment of productive producer cells.

AB - With the growing demand for therapeutic proteins such as antibodies or vaccines, manufactures are in need to deliver products at high quality and constant productivity. For the biotechnological production of these products, mammalian cells are predominantly used. The productivity of producer cells is, amongst others, dependent on the number of integrated transgene copies per cell, and thus elevating stable gene transfer efficiencies to ensure sustained expression of the gene of interest is paramount. In this study the Sleeping Beauty transposon two-component vector system, consisting of a transposon donor plasmid and a transposase expression plasmid, was used to generate stable cell pools. However, using sensitive genomic PCR and reverse transcriptase PCR (RT-PCR), the transposase gene integration and expression was demonstrated for a time period of 48 days post transfection. To provide an alternative to the employment of plasmid-based transposase expression circumventing potential re-mobilization events of the already stably transposed transgenes, the transposase gene was transcribed in vitro into mRNA. After co-transfection of transposase transcripts at different ratios to the donor vector efficient transposition was obtained. This study demonstrated that this technical approach mediated high copy numbers and expression levels of transgenes in recombinant cells without the risk of undesired extended transposase expression. Besides transposons, retroviral vectors are frequently used to introduce foreign genetic material into mammalian cells aiming for the establishment of producer cells. Such viral vectors are commonly pseudotyped with VSV-G achieving high vector copy numbers in mammalian cells. However, this requires handling under BSL-2 conditions. To circumvent this, viral vectors were equipped with the ecotropic envelope protein PVC211mc, a molecular clone of Friend murine leukemia virus (MLV), enabling transduction of CHO cells and murine hematopoietic stem cells but allowing experiments in BSL- 1 laboratories. The aim of this work was to optimize gene transfer efficiencies by generating PVC211-derived envelope protein (Env) variants lacking the R-peptide and thus rendering the Env proteins fusogenic. All generated variants failed to efficiently pseudotype MLV vectors but two variants successfully pseudotyped HIV-1 particles. The HIV-1 vectors pseudotyped with the envelope variant eMLV-GaLVΔR mediated superior infectivity as compared to wild-type Env. HIV(eMLV-GaLVΔR) should prove useful as a tool for the establishment of productive producer cells.

U2 - 10.15488/15372

DO - 10.15488/15372

M3 - Doctoral thesis

CY - Hannover

ER -