Scalable Production of AAV Vectors in Orbitally Shaken HEK293 Cells

Adeno-associated virus (AAV) vectors are currently among the most commonly applied for in vivo gene therapy approaches. The evaluation of vectors during clinical development requires the production of considerable amounts of highly pure and potent vectors. Here, we set up a scalable process for AAV production, using orbitally shaken bioreactors and a fully characterized suspension-adapted cell line, HEKExpress. We conducted a proof-of-concept production of AAV2/8 and AAV2/9 vectors using HEKExpress cells. Furthermore, we compared the production of AAV2/9 vectors using this suspension cell line to classical protocols based on adherent HEK293 cells to demonstrate bioequivalence in vitro and in vivo. Following upstream processing, we purified vectors via gradient centrifugation and immunoaffinity chromatography. The in vitro characterization revealed differences due to the purification method, as well as the transfection protocol and the corresponding HEK293 cell line. The purification method and cell line used also affected in vivo transduction efficiency after bilateral injection of AAV2/9 vectors expressing a GFP reporter fused with a nuclear localization signal (AAV2/9-CBA-nlsGFP) into the striatum of adult mice. These results show that AAV vectors deriving from suspension HEKExpress cells are bioequivalent and may exhibit higher potency than vectors produced with adherent HEK293 cells.

Improved biocompatibility of polyethylenimine (PEI) as a gene carrier by conjugating urocanic acid: In vitro and in vivo

Jeffrey Alan Hubbell, Jong Eun Ihm, Irina Krier

Polyethylenimine (PEI) is a promising gene carrier among polymeric vectors because of its high transfection efficiency, buffering capacity, which is responsible for its escape from endosomes, facility of modification, reasonable price and wide range of molecular weight. However, toxicity of PEI is an important obstacle to overcome for using it as a gene carrier in gene therapy protocols. In our study, PEI (10 kDa) was modified with urocanic acid, which has an imidazole ring (PEIU). This modification of PEI played an important role by improving transfection efficiency and reducing toxicity. The PEIU was condensed with DNA in different nitrogen of polycation/phosphate of DNA (N/P) ratios starting from 1.25. The PEIU/DNA complexes formed smaller (60-80 versus 100-400 nm) and less dispersible particles than PEI/DNA complexes. Cell viability test showed that in all the transfection experiments PEIU was always found less toxic than the PEI on three different cell-lines, Cos-7, HeLa, and 293T. The PEIU demonstrated improved biocompatibility as compared to the PEI. The in vitro transfection experiments showed that the PEIU displayed similar or lower transfection efficiencies than the PEI in the absence of serum. In medium complemented with serum, the PEIU had higher transfection efficiencies than the PEI. In vivo transfection experiments were carried out with intravenous and subcutaneous injections into mice. Five different organs were taken and lysed for quantification of expressed GFP. The quantification of the fluorescence of these organs exhibited that the PEIU has better transfection efficiency in in vivo experiments compared to the PEI. This study generated a strong evidence indicating that PEIU can be considered as a promising versatile gene carrier because of its biocompatibility and good transfection efficiency.

Polymer Soc Korea2015

Lentiviral Vector-Mediated Gene Delivery for the Generation of Chinese Hamster Ovary Cell Lines with Improved Productivity and Stability

Agata Oberbek

Recombinant cell line generation by standard transfection techniques is a time-consuming and labor-intensive process often leading to an unpredictable outcome as transgene integration is a rare, random event. Consequently, the population of cells obtained after standard gene delivery is heterogeneous in terms of specific growth and recombinant protein productivity. Therefore, genetic selection is usually applied to the population for up to 3 weeks to eliminate non-recombinant cells. A large number of the surviving cell lines must be screened to find a few which stably produce the recombinant protein at the desired level. These must be maintained in culture for 2-4 months in the absence of selection to assess the stability of protein production. In contrast, lentiviral vectors (LVs) deliver the recombinant gene to target cells through infection and a controlled integration process, which allows the inserted transgene to become a permanent genetic element of the transduced cell. Moreover, lentiviruses tend to integrate into transcriptionally active sites of the host cell genome. Hence, LV-mediated gene transfer was expected to increase the probability of generating a stable high-producing clone in comparison to the standard methods based on plasmid transfection. Therefore, the objective of this PhD thesis was to improve the efficiency of stable cell line generation with the use of LVs. For this purpose, LVs carrying the enhanced green fluorescent protein (eGFP) gene under the control of either the cytomegalovirus immediate early promoter/enhancer (CMV) or the elongation factor-1 alpha promoter (EF-1α) were generated and used to infect Chinese hamster ovary (CHO) cells. Two days after gene delivery at a multiplicity of infection (MOI) of 2, up to 98% of infected cells were eGFP-positive. Following isolation by fluorescence-activated cell sorting (FACS), CHO cell pools and clonal cell lines were analyzed for the stability of eGFP expression over time. In these studies, the EF-1α promoter was found to yield more stable and homogeneous protein expression compared to the CMV promoter. For comparison, cell pools and cell lines were generated by transfection with the LV transfer plasmid bearing the eGFP gene. The level and stability of eGFP expression was greater in LV-generated pools and cell lines than in those established by transfection. Subsequently, an LV expressing the tumor necrosis factor receptor-Fc (TNFR-Fc) fusion gene and the eGFP gene from a bicistronic mRNA under the control of the EF-1α promoter (LV_EF-TNFR) was generated and used to infect CHO cells. Based on selective FACS gating for high eGFP expression, stable CHO clones showing volumetric productivities of up to 100 mg/L of TNFR-Fc in a non-optimized batch process could be isolated within 2 days of infection at an MOI of 2. The level of expression of the recombinant protein remained constant for 3 months in serum-free suspension culture and in the absence of chemical selective agents. Next, CHO cells were infected at 3 higher MOIs with the LV_EF-TNFR and clonal cell lines with high eGFP-specific fluorescence were recovered by FACS at 2 d post-infection. These cell lines had volumetric TNFR:Fc productivities ranging from 100 to 400 mg/L in 4-day cultures with cell-specific secretion rates up to 100 pg/cell/day. Moreover, recombinant cell lines developed by the LV-based technology could be successfully cultured in bench-scale bioreactors, yielding up to 1.5 g/L of TNFR:Fc under batch mode operation. The increase in integrated TNFR:Fc copy number was found to correlate with the MOI, and resulted in improved protein productivity. However, this relationship was found to be non-linear: cell saturation in terms of protein productivity and integrated transgene copy number was observed at an MOI of 100. In order to determine the shortest time-frame necessary for the production of satisfactory quantities of a recombinant product, the high MOI-infection approach was tested on pools of transduced cells. Without a priori FACS isolation, recombinant pools of CHO cells expressing on average 500 mg/L of TNFR:Fc within 8 days of inoculation at 3 × 105 cells/ml could be obtained. This strategy provided a simple protocol for high titer recombinant protein production, executable within 4 weeks from LV batch preparation to product harvest. Since large-scale production of LVs would be needed for industrialized bioprocesses, a method for LV production in serum-free suspension culture was developed. However, the best titers generated by this system were still approximately 20-fold lower than the yields obtained regularly from the adherent cultures, thus leaving room for further optimization. In conclusion, LV-mediated gene transfer provided an efficient alternative to plasmid transfection for the generation of stable and high-producing recombinant cell lines. Moreover, it proved amenable to large-scale manufacture and could yield several hundred milligrams per liter of the recombinant product without the need for any kind of genetic selection. In addition, using the LV technology described here it seems possible to target the number of copies of the delivered transgene to a specific range which balances favorable growth with good productivity characteristics.

EPFL2010

Transgene expression in the guinea pig cochlea mediated by a lentivirus-derived gene transfer vector

Didier Trono, Romain Zufferey

The utility of lentivirus as a gene delivery vector in the cochlea was evaluated in vitro and in vivo. Lentivirus transduction was assessed through expression analysis of a reporter gene, green fluorescent protein (GFP), integrated within the viral genome. In vitro characterization of lentivirus-GFP was assessed by infection of explants from cochleas of neonatal rat. The lentiviral vector transduced both spiral ganglion neurons (SGNs) and glial cells. In vivo characterization of lentivirus-GFP was assessed by directly infusing the vector into the guinea pig cochlea via an osmotic minipump. Sections of lentivirus-infused cochlea revealed a highly restricted fluorescence pattern limited to the periphery of the perilymphatic space. Transduction of SGNs and glial cells by lentivirus in vitro but not in vivo suggests limited dissemination of the viral vector from the perilymphatic space. The cellular and tissue architecture of the lentivirus-infused cochlea was intact and free of inflammation. Restricted transduction of cell types confined to the periphery of the perilymphatic space by the lentivirus is ideal for stable production of gene products secreted into the perilymph.