Inhibition of glycogen synthase kinase-3 enhances the differentiation and reduces the proliferation of adult human olfactory epithelium neural precursors.

The olfactory epithelium (OE) contains neural precursor cells which can be easily harvested from a minimally invasive nasal biopsy, making them a valuable cell source to study human neural cell lineages in health and disease. Glycogen synthase kinase-3 (GSK-3) has been implicated in the etiology and treatment of neuropsychiatric disorders and also in the regulation of murine neural precursor cell fate in vitro and in vivo. In this study, we examined the impact of decreased GSK-3 activity on the fate of adult human OE neural precursors in vitro. GSK-3 inhibition was achieved using ATP-competitive (6-bromoindirubin-3'-oxime and CHIR99021) or substrate-competitive (TAT-eIF2B) inhibitors to eliminate potential confounding effects on cell fate due to off-target kinase inhibition. GSK-3 inhibitors decreased the number of neural precursor cells in OE cell cultures through a reduction in proliferation. Decreased proliferation was not associated with a reduction in cell survival but was accompanied by a reduction in nestin expression and a substantial increase in the expression of the neuronal differentiation markers MAP1B and neurofilament (NF-M) after 10 days in culture. Taken together, these results suggest that GSK-3 inhibition promotes the early stages of neuronal differentiation in cultures of adult human neural precursors and provide insights into the mechanisms by which alterations in GSK-3 signaling affect adult human neurogenesis, a cellular process strongly suspected to play a role in the etiology of neuropsychiatric disorders.

Selective enhancement of the uptake and bioactivity of a TAT-conjugated peptide inhibitor of glycogen synthase kinase-3.

The use of cell-penetrating peptides as transduction vectors is a promising approach to deliver peptides and proteins into cells. However, the uptake and bioavailability of trans-activating transcriptor (TAT)-conjugated molecules vary depending on the cell type and the cargo. This study aimed to determine whether a low-voltage electrical pulse can enhance the TAT-mediated delivery of peptide cargoes in different cell types. In TF-1 and mouse embryonic stem cells, the uptake of a novel detachable TAT-conjugated glycogen synthase kinase-3 (GSK-3) peptide inhibitor was enhanced by an order of magnitude without affecting the cell viability. A similar increase in uptake was achieved in primary mouse bone marrow cells while maintaining >80% of their viability. Interestingly, under these low-voltage conditions, the uptake of a control peptide not conjugated to TAT was not significantly increased. A T-cell factor/lymphoid enhancer factor (TCF/LEF) luciferase reporter assay was also used to assess the bioactivity of the TAT construct. The results indicated that cells loaded with a low-voltage electrical pulse had a twofold increase in TCF/LEF activity, which was equivalent to a level of GSK-3 inhibition similar to that of cells treated with 20 mmol/l lithium or 500 nmol/l (2'Z,3'E)-6-bromoindirubin-3'-oxime. These results demonstrate the usefulness of low-voltage electrical pulses to enhance the uptake and bioactivity of TAT-conjugated molecules in different cell types.

Measurement of cell-penetrating peptide-mediated transduction of adult hematopoietic stem cells.

The ability of cell-penetrating peptides (CPPs) to cross cell membranes and transport cargo into cells makes them an attractive tool for the molecular engineering of stem cells. Even though the exact mechanism of transduction remains unclear, their potential has been demonstrated for diverse applications, including hematopoietic stem cell expansion and the generation of islets cells from embryonic stem cells. Several parameters can affect the intracellular delivery of CPP-based constructs. Those include the type of cells targeted, the type of CPP used, and the properties of the cargo. For this reason, it is important to have a means to quantitatively assess the transduction efficiency of specific constructs in the cell type of interest in order to select the best vector for a specific application. In this chapter, we describe a method to measure the uptake of HIV transactivator of transcription (TAT) and the homeobox protein Antennapedia (Antp) constructs in primary hematopoietic progenitor cells and hematopoietic cell lines. This method is useful to compare, select, and optimize different strategies to deliver CPP-based constructs into a given cell type.

Scalable Lentiviral Vector Production Using Stable HEK293SF Producer Cell Lines.

Lentiviral vectors (LV) represent a key tool for gene and cell therapy applications. The production of these vectors in sufficient quantities for clinical applications remains a hurdle, prompting the field toward developing suspension processes that are conducive to large-scale production. This study describes a LV production strategy using a stable inducible producer cell line. The HEK293 cell line employed grows in suspension, thus offering direct scalability, and produces a green fluorescent protein (GFP)-expressing lentiviral vector in the 106 transduction units (TU)/mL range without optimization. The stable producer cell line, called clone 92, was derived by stable transfection from a packaging cell line with a plasmid encoding the transgene GFP. The packaging cell line expresses all the other necessary components to produce LV upon induction with cumate and doxycycline. First, the study demonstrated that LV production using clone 92 is scalable from 20 mL shake flasks to 3 L bioreactors. Next, two strategies were developed for high-yield LV production in perfusion mode using acoustic cell filter technology in 1-3 L bioreactors. The first approach uses a basal commercial medium and perfusion mode both pre- and post-induction for increasing cell density and LV recovery. The second approach makes use of a fortified medium formulation to achieve target cell density for induction in batch mode, followed by perfusion mode after induction. Using these perfusion-based strategies, the titer was improved to 3.2 × 107 TU/mL. As a result, cumulative functional LV titers were increased by up to 15-fold compared to batch mode, reaching a cumulative total yield of 8 × 1010 TU/L of bioreactor culture. This approach is easily amenable to large-scale production and commercial manufacturing.

Accelerated mass production of influenza virus seed stocks in HEK-293 suspension cell cultures by reverse genetics.

Despite major advances in developing capacities and alternative technologies to egg-based production of influenza vaccines, responsiveness to an influenza pandemic threat is limited by the time it takes to generate a Candidate Vaccine Virus (CVV) as reported by the 2015 WHO Informal Consultation report titled "Influenza Vaccine Response during the Start of a Pandemic". In previous work, we have shown that HEK-293 cell culture in suspension and serum free medium is an efficient production platform for cell culture manufacturing of influenza candidate vaccines. This report, took advantage of, recombinant DNA technology using Reverse Genetics of influenza strains, and advances in the large-scale transfection of suspension cultured HEK-293 cells. We demonstrate the efficient generation of H1N1 with the PR8 backbone reassortant under controlled bioreactor conditions in two sequential steps (transfection/rescue and infection/production). This approach could deliver a CVV for influenza vaccine manufacturing within two-weeks, starting from HA and NA pandemic sequences. Furthermore, the scalability of the transfection technology combined with the HEK-293 platform has been extensively demonstrated at >100L scale for several biologics, including recombinant viruses. Thus, this innovative approach is better suited to rationally engineer and mass produce influenza CVV within significantly shorter timelines to enable an effective global response in pandemic situations.

Generation of monoclonal pan-hemagglutinin antibodies for the quantification of multiple strains of influenza.

Vaccination is the most effective course of action to prevent influenza. About 150 million doses of influenza vaccines were distributed for the 2015-2016 season in the USA alone according to the Centers for Disease Control and Prevention. Vaccine dosage is calculated based on the concentration of hemagglutinin (HA), the main surface glycoprotein expressed by influenza which varies from strain to strain. Therefore yearly-updated strain-specific antibodies and calibrating antigens are required. Preparing these quantification reagents can take up to three months and significantly slows down the release of new vaccine lots. Therefore, to circumvent the need for strain-specific sera, two anti-HA monoclonal antibodies (mAbs) against a highly conserved sequence have been produced by immunizing mice with a novel peptide-conjugate. Immunoblots demonstrate that 40 strains of influenza encompassing HA subtypes H1 to H13, as well as B strains from the Yamagata and Victoria lineage were detected when the two mAbs are combined to from a pan-HA mAb cocktail. Quantification using this pan-HA mAbs cocktail was achieved in a dot blot assay and results correlated with concentrations measured in a hemagglutination assay with a coefficient of correlation of 0.80. A competitive ELISA was also optimised with purified viral-like particles. Regardless of the quantification method used, pan-HA antibodies can be employed to accelerate process development when strain-specific antibodies are not available, and represent a valuable tool in case of pandemics. These antibodies were also expressed in CHO cells to facilitate large-scale production using bioreactor technologies which might be required to meet industrial needs for quantification reagents. Finally, a simulation model was created to predict the binding affinity of the two anti-HA antibodies to the amino acids composing the highly conserved epitope; different probabilities of interaction between a given amino acid and the antibodies might explain the affinity of each antibody against different influenza strains.

Critical review of current and emerging quantification methods for the development of influenza vaccine candidates.

Significant improvements in production and purification have been achieved since the first approved influenza vaccines were administered 75 years ago. Global surveillance and fast response have limited the impact of the last pandemic in 2009. In case of another pandemic, vaccines can be generated within three weeks with certain platforms. However, our Achilles heel is at the quantification level. Production of reagents for the quantification of new vaccines using the SRID, the main method formally approved by regulatory bodies, requires two to three months. The impact of such delays can be tragic for vulnerable populations. Therefore, efforts have been directed toward developing alternative quantification methods, which are sensitive, accurate, easy to implement and independent of the availability of specific reagents. The use of newly-developed antibodies against a conserved region of hemagglutinin (HA), a surface protein of influenza, holds great promises as they are able to recognize multiple subtypes of influenza; these new antibodies could be used in immunoassays such as ELISA and slot-blot analysis. HA concentration can also be determined using reversed-phase high performance liquid chromatography (RP-HPLC), which obviates the need for antibodies but still requires a reference standard. The number of viral particles can be evaluated using ion-exchange HPLC and techniques based on flow cytometry principles, but non-viral vesicles have to be taken into account with cellular production platforms. As new production systems are optimized, new quantification methods that are adapted to the type of vaccine produced are required. The nature of these new-generation vaccines might dictate which quantification method to use. In all cases, an alternative method will have to be validated against the current SRID assay. A consensus among the scientific community would have to be reached so that the adoption of new quantification methods would be harmonized between international laboratories.

Particle quantification of influenza viruses by high performance liquid chromatography.

The influenza virus continuously undergoes antigenic evolution requiring manufacturing, validation and release of new seasonal vaccine lots to match new circulating strains. Although current production processes are well established for manufacturing seasonal inactivated influenza vaccines, significant limitations have been underlined in the case of pandemic outbreaks. The World Health Organization called for a global pandemic influenza vaccine action plan including the development of new technologies. A rapid and reliable method for the quantification of influenza total particles is crucially needed to support the development, improvement and validation of novel influenza vaccine manufacturing platforms. This work presents the development of an ion exchange-high performance liquid chromatography method for the quantification of influenza virus particles. The method was developed using sucrose cushion purified influenza viruses A and B produced in HEK 293 suspension cell cultures. The virus was eluted in 1.5 M NaCl salt with 20 mM Tris-HCl and 0.01% Zwittergent at pH 8.0. It was detected by native fluorescence and the total analysis time was 13.5 min. A linear response range was established between 1 × 10(9) and 1 × 10(11) virus particle per ml (VP/ml) with a correlation coefficient greater than 0.99. The limit of detection was between 2.07 × 10(8) and 4.35 × 10(9) whereas the limit of quantification was between 6.90 × 10(8) and 1.45 × 10(10)VP/ml, respectively. The coefficient of variation of the intra- and inter-day precision of the method was less than 5% and 10%. HPLC data compared well with results obtained by electron microscopy, HA assay and with a virus counter, and was used to monitor virus concentrations in the supernatant obtained directly from the cell culture production vessels. The HPLC influenza virus analytical method can potentially be suitable as an in-process monitoring tool to accelerate the development of processes for the manufacturing of influenza vaccines.