LentiPro stable producer cells: Delivering scalable and reliable lentiviral vector manufacturing

Lentiviral vectors are one of the most currently used viral vectors for gene and cell therapies. Their use in clinical protocols has significantly increased in the past 5 years with the approval of several gene therapeutic products relying on lentiviral vector gene delivery. Capable of transducing non-dividing cells and presenting safer integration profiles as self-inactivating vectors, lentiviral vectors have progressively undertaken gammaretroviral vector use in gene therapies. However the knowledge on lentiviral vector manufacture is far more immature than that of gammaretroviral vectors. While the production of gammaretrovirus rely on stable producer cell lines and perfusion systems, enabling high cell density and longer term productions, most of the bioprocesses for lentiviral bioproducts rely on transient transfections and short term batch productions. At the upstream process, many of the challenges lentiviral bioproducts present in their manufacturing are related to the apoptosis leading cytotoxicity of some of the vector components. Supported on our long track experience and enabling tools developed for gammaretrovirus manufacturing, we carried out the challenge of establishing a constitutive stable lentiviral producer cell line. To surpass the challenges we proposed to eliminate or reduce the cytotoxicity of the lentiviral vector expression components1. Several strategic novelties were introduced in the development of the cell line namely: (i) the use of a modified gag-pro-pol, (ii) introduction of all the third generation lentiviral expression cassettes by chemical transfection instead of viral transduction and (iii) performing only one clone screening step (enabling the use on the ‘Single step cloning screening’ protocol developed by our group2). After establishing a stable producer cell line the culture conditions were developed with the main aim of extending bioreaction culture time and viral vector total yields. A lentiviral producer cell line constitutively producing infective titers above 106 TU.mL-1.day-1 was established. Moreover the new protocol to generate the cell line enabled its development in less than six months. The cell line showed to be stable, consistently maintaining vector productivity over one month in the absence of antibiotics. At the bioreaction process it was possible to maintain the cells continuously producing over 10 days1. These results validate the transition to continuous or perfusion large-scale production systems qualifying the strengths and advantages of the strategies followed. This work to be presented will discuss the challenges on the manufacture and scale-up of lentiviral vectors as well the strategies and novel technologies to be adopted to enable effective upstream processes. 1Tomás et al. (2018) ‘LentiPro26: novel stable cell lines for constitutive lentiviral vector production\u27 Sci Rep. 8(1):5271 2 Rodrigues et al. (2015) ‘Single step cloning-screening method: a new tool for developing and studying high-titer viral vector producer cells\u27 Gene Ther. 22(9):6

Pseudotyping retrovirus like particles vaccine candidates with Hepatitis C virus envelope protein E2 requires the cellular expression of CD81

Hepatitis C virus (HCV) infects 3% of world population being responsible for nearly half a million deaths annually urging the need for a prophylactic vaccine. Retrovirus like particles are commonly used scaffolds for antigens presentation being the core of diverse vaccine candidates. The immunogenicity of host proteins naturally incorporated in retrovirus was hypothesized to impact the performance of retrovirus based vaccines. In this work, the capacity of engineered retrovirus like particles devoided of host protein CD81 to display HCV envelope antigens was compared to non-engineered particles. A persistent inability of CD81 negative VLPs to incorporate HCV E2 protein as a result from the inefficient transport of HCV E2 to the plasma membrane, was observed. This work enabled the identification of a CD81-mediated transport of HCV E2 while stressing the importance of host proteins for the development of recombinant vaccines.publishe

Evaluation of Structurally Distorted Split GFP Fluorescent Sensors for Cell-Based Detection of Viral Proteolytic Activity

Cell-based assays are essential for virus functional characterization in fundamental and applied research. Overcoming the limitations of virus-labelling strategies while allowing functional assessment of critical viral enzymes, virus-induced cell-based biosensors constitute a powerful approach. Herein, we designed and characterized different cell-based switch-on split GFP sensors reporting viral proteolytic activity and virus infection. Crucial to these sensors is the effective—yet reversible—fluorescence off-state, through protein distortion. For that, single (protein embedment or intein-mediated cyclization) or dual (coiled-coils) distortion schemes prevent split GFP self-assembly, until virus-promoted proteolysis of a cleavable sequence. All strategies showed their applicability in detecting viral proteolysis, although with different efficiencies depending on the protease. While for tobacco etch virus protease the best performing sensor was based on coiled-coils (signal-to-noise ratio, SNR, 97), for adenovirus and lentivirus proteases it was based on GFP11 cyclization (SNR 3.5) or GFP11 embedment distortion (SNR 6.0), respectively. When stably expressed, the sensors allowed live cell biosensing of adenovirus infection, with sensor fluorescence activation 24 h post-infection. The structural distortions herein studied are highly valuable in the development of cellular biosensing platforms. Additionally highlighted, selection of the best performing strategy is highly dependent on the unique properties of each viral protease

Cell specific productivity of HD CAVGFP and corresponding contamination with helper JBΔ5.

<p>Errors correspond to 25% inter-assay variability. The contamination levels of JBΔ5 correspond to the ratio between the I.P titers of JBΔ5 and HD CAVGFP.</p

Bioactive transparent films based on polysaccharides and cholinium carboxylate ionic liquids

Novel antibacterial and biocompatible transparent films based on chitosan or pullulan and two bioactive ionic liquids (ILs), cholinium hexanoate and cholinium citrate, were prepared. These ILs were selected based on their MIC values against several microbial strains, film-forming ability when blended with the polysaccharides and biocompatibility against designated human cell lines. The films were obtained through simple casting of polysaccharide aqueous solutions containing different amounts of the ILs (20 and 40 wt% with respect to the amount of polysaccharide). The physical properties of the films were investigated using transmittance measurements, thermal analysis, mechanical testing and antibacterial assays. In general, the addition of both ILs does not affect the optical transparency (up to 80% transmittance within 400-700 nm) of the films but decreased their stiffness (acting as plasticizers) and thermal stability. All chitosan-based films showed antibacterial activity against S. aureus and K. pneumoniae but for pullulan only those with cholinium citrate were bioactive

Cell specific viral titers obtained for MDCK-E1 cell clones and the control cell DKZeo at different cell culture passages, in order to assess its effect on the production of CAVGFP.

<p>Low passage corresponds to passage number 20, intermediate to passages between 30–40 and high passage to passages 45–55. Error bars represent a 25% inter-assay variability.</p