301. Efficient Delivery of Nuclear and Cytoplasmic Proteins Fused to HIV-1 Gag Polypeptide By Means of Virus-Like Particles

The expression of the retroviral polypeptide Gag can induce the formation of virus-like particles (VLP). Upon expression, the polypeptide is targeted to the cell membrane and incorporated in the VLP during membrane budding. Chimeric proteins consisting of Gag polypeptide fused to different proteins were engineered and the VLP produced were used as vehicles for protein delivery. One major advantage of this approach is the low mutation risk due to the absence of DNA transfer and genomic integration. In this study, the C-Terminus of Gag from HIV-1 was fused to the green fluorescent protein (GFP), a chimeric transactivator (cTA) and a reprogamming factor (KLF4). VLP were produced by transfection using a stable cell line (293SF-pacLV) that expresses VSVg and Gag-pol. Analysis of the supernatants from producing cells by western blot confirmed the presence of Gag-GFP, -cTA and -KLF4. Confocal microscopy showed that the vast majority of the cells (> 90%) treated with VLP-GFP/polybrene complexes was successfully transduced. The cells also displayed a GFP signal almost exclusively localized inside the cytoplasm. Additional VSVg expression during the production facilitated the endosomal escape of VLP-GFP in transduced cells. The insertion of a nuclear localisation signal (NLS) shifted the localization of the GFP to the cell nucleus demonstrating that a nuclear protein could be successfully delivered by VLP. The experiment was thus repeated using two transcription factors. Lentiviral vectors were used to make two stable pools of HEK293 cells each containing a specific GFP reporter cassette. The GFP gene was regulated either by the CR5 promoter (specifically activated by the cTA) or by a minimal promoter fused to KLF4 transcription response elements (TRE). Transduction of the CR5-GFP pool with VLP-cTA/polybrene complexes showed a powerful activation of the reporter (365-fold compared to the negative control) as measured by flow cell cytometry two days post-transduction. Surprisingly, no activation was observed in the TRE-GFP pool three days after transduction by VLP-KLF4/polybrene complexes. Evidence obtained by transfection suggested that the Gag fusion inhibits KLF4 activity. To augment activity, the activation domain of VP16 was fused to KLF4. Transfection of a plasmid encoding Gag-VP16KLF4 strongly activated the reporter by a factor of 126-fold (6-fold higher than wild type KLF4). The ability of VLP produced with Gag-VP16KLF4 to activate transcription is currently under investigation. In summary, VLP based on HIV-1 Gag can deliver nuclear and cytoplasmic proteins directly into cells with a low mutation risk. Therefore, our platform for VLP production could be useful for several applications including cell reprogramming and genome editing oriented toward cell therapies of diseases like Duchenne muscular dystrophy

555. Development of a Post-Exposure Treatment for Ebola Virus Infections Based on AAV Vectors and Zmapp Antibody Cocktail

The recent Ebola outbreak in West Africa has been the deadliest in the history. To prevent future recurrence of such outbreak, better treatments and effective vaccines against Ebola virus are desirable. Among such promising treatments, the Zmapp cocktail containing neutralizing antibodies (13C6, 2G4 and 4G7) has successfully treated some patients. However, the feasibility of using it on large populations especially in developing countries is questionable. To address this potential issue, we propose to employ recombinant vectors derived from adeno-associated virus (rAAV). There are several advantages of using rAAV: because of 1) their safety profile; 2) only one injection (or a few) would be required; 3) the high stability of lyophilized rAAVs at ambient temperature and; 4) the panel of available serotypes. Because of these interesting features, we are currently developing a treatment based on three rAAVs to deliver the genes for the Zmapp cocktail of antibodies. We have already produced at small scale a rAAV expressing the 2G4 antibody. The DNA sequences for the heavy chain and light chains were codon-optimized for better expression in humans and were designed to be expressed from the same gene. A strong promoter (CAG) resistant to silencing in vivo was chosen to drive gene expression of the antibody. The rAAV were produced by transfection using our patented cGMP compatible HEK293 cell line. The production was performed in suspension culture in the absence of serum. Secretion of 2G4 antibody by rAAV transduced cells (HEK293 and CHO cells) was confirmed. The results demonstrated that rAAV-CAG-2G4 was functional and allowed for the correct assembly of the heavy and light chains of 2G4. Purification of 200 mL of rAAV-CAG-2G4 production was performed by ultracentrifugation on an iodixanol density-step gradient. Two other rAAVs coding 13C6 and 4G7 antibodies are in the processed of being constructed and produced in a similar manner. We are also in the process of comparing the efficacy of two serotypes of AAV (9 and DJ) in mice by intranasal delivery. Using the best serotype, the rAAVs will be produced and purified from a starting suspension culture of 20 L. Their efficacy for treating Ebola infections will then be evaluated in a mouse model infected by the virus

708. Process Development of Lentiviral Vectors for Large Scale Production Using a HEK293 Producer Cell Line

Lentiviral vectors (LVs) are promising vectors for gene therapy. Most often, they are used to deliver a functional copy of a gene to sustainably replace a defective or missing gene. However, processes for LVs must be improved to increase the yield, facilitate the scale up and satisfy Health regulatory agencies. For these reasons, we have developed and optimized a LVs production process in serum-free medium using an inducible HEK293 producer cell line which possesses the capacity to grow in suspension culture. By adding two inducing molecules, (cumate and doxycycline) this cell line produces LVs pseudotyped with the protein G of the vesicular stomatitis virus without the need of any transfection. Our tested LV carried an expression cassette for GFP to facilitate LV quantification. To optimize the process, a design of experiment (DoE) was prepared which included the study of different culture media, high cell density production using six cell boosts commercially available and the addition of sodium butyrate, caffeine and valproic acid. We found that two cell boosts were outperforming the other cell boosts tested. At the present time, two commercial media (Hycell TransFx-H and SFM4TransFx-293 media) were our best candidates to maximize viral titer by achieving high cell density culture. In parallel, a LV carrying the cDNA for a shorter version of dystrophin (mini-dystrophin) was constructed. The truncated version of the dystrophin was produced by transient transfection in 293A cells and its presence was confirmed by western blot. We are planning to evaluate if the optimal conditions for the production of LV-GFP will be also applicable to LV-mini-dystrophin, a LV encoding a much longer transgene than GFP (0.7 kb vs 5.8 kb). This LV could be first evaluated for cell therapy in animal models and later, in patients suffering from Duchenne muscular dystrophy, where the dystrophin gene is defective and the protein is absent

Fluorescent labeling in semi-solid medium for selection of mammalian cells secreting high-levels of recombinant proteins

<p>Abstract</p> <p>Background</p> <p>Despite the powerful impact in recent years of gene expression markers like the green fluorescent protein (GFP) to link the expression of recombinant protein for selection of high producers, there is a strong incentive to develop rapid and efficient methods for isolating mammalian cell clones secreting high levels of marker-free recombinant proteins. Recently, a method combining cell colony growth in methylcellulose-based medium with detection by a fluorescently labeled secondary antibody or antigen has shown promise for the selection of Chinese Hamster Ovary (CHO) cell lines secreting recombinant antibodies. Here we report an extension of this method referred to as fluorescent labeling in semi-solid medium (FLSSM) to detect recombinant proteins significantly smaller than antibodies, such as IGF-E5, a 25 kDa insulin-like growth factor derivative.</p> <p>Results</p> <p>CHO cell clones, expressing 300 μg/ml IGF-E5 in batch culture, were isolated more easily and quickly compared to the classic limiting dilution method. The intensity of the detected fluorescent signal was found to be proportional to the amount of IGF-E5 secreted, thus allowing the highest producers in the population to be identified and picked. CHO clones producing up to 9.5 μg/ml of Tissue-Plasminogen Activator (tPA, 67 kDa) were also generated using FLSSM. In addition, IGF-E5 high-producers were isolated from 293SF transfectants, showing that cell selection in semi-solid medium is not limited to CHO and lymphoid cells. The best positive clones were collected with a micromanipulator as well as with an automated colony picker, thus demonstrating the method's high throughput potential.</p> <p>Conclusion</p> <p>FLSSM allows rapid visualization of the high secretors from transfected pools prior to picking, thus eliminating the tedious task of screening a high number of cell isolates. Because of its rapidity and its simplicity, FLSSM is a versatile method for the screening of high producers for research and industry.</p

321. Deletion of Mutated GAA Repeats from the Intron 1 of the Frataxin Gene Using the CRISPR System Restores the Protein Expression in a Friedreich Ataxia Model

The CRISPR system is now widely used as a molecular tool to edit the genome. We used this technique in Friedreich Ataxia (FRDA), an inherited autosomal disease known to cause a decrease of the mitochondrial frataxin protein. Genetic analysis revealed a GAA repeat expansion within the intron 1 of the frataxin (FXN) gene. We used cells derived from the YG8sR mouse model where the mouse frataxin gene is knockout but contain a human FXN mutated transgene on one allele. We then deleted the GAA trinucleotide repeat using 2 specific guide RNAs (gRNAs) co-expressed with either S. pyogenes (Sp) or S. aureus (Sa) Cas9. We were able to monitored an increase up to 2-fold of frataxin mRNA and protein levels in clone cells. We also confirmed these results in vivo using DNA electroporation in the Tibialis anterior muscle of the YG8R mice. Ongoing in vivo investigation of a systemically injected AAV-DJ vector expressing the SaCas9 and 2 successful selected gRNAs in the mouse model YG8sR will hopefully provide more details answers on the efficacy of the approach and give us preliminary data to go forward for clinical trial. The deletion of the GAA repeats expansion then might be a highly valuable gene therapy approach for FRDA patients

Evaluation of novel HIV vaccine candidates using recombinant vesicular stomatitis virus vector produced in serum-free Vero cell cultures.

Acquired Immune Deficiency Syndrome (AIDS) in humans is a result of the destruction of the immune system caused by Human Immunodeficiency Virus (HIV) infection. This serious epidemic is still progressing world-wide. Despite advances in treatment, a safe and effective preventive HIV vaccine is desired to combat this disease, and to save millions of lives. However, such a vaccine is not available yet although extensive amounts of resources in research and development have been invested over three decades. In light of the recently approved Ebola virus disease vaccine based on a recombinant vesicular stomatitis virus (rVSV-ZEBOV), we present the results of our work on three novel VSV-vectored HIV vaccine candidates. We describe the design, rescue, production and purification method and evaluate their immunogenicity in mice prior to preclinical studies that will be performed in non-human primates. The production of each of the three candidate vaccines (rVSV-B6-NL4.3Env/SIVtm, rVSV-B6-NL4.3Env/Ebtm and rVSV-B6-A74Env(PN6)/SIVtm) was evaluated in small scale in Vero cells and it was found that production kinetics on Vero cells vary depending on the HIV gp surface protein used. Purified virus preparations complied with the WHO restrictions for the residual DNA and host cell protein contents. Finally, when administered to mice, all three rVSV-HIV vaccine candidates induced an HIV gp140-specific antibody response

Technological developments as an answer to bridge management challenges

IABSE Symposium 2019, Towards a Resilient Built Environment - Risk and Asset Management, GUIMARAES, PORTUGAL, 27-/03/2019 - 29/03/2019Bridge management is a challenge as owners have to deal with limited financial resources to maintain the functionalities and safety of ageing structures. Demands on transportation networks change, due to regulatory developments, society&apos;s evolution and shifts with high expectations on the operational performance of roadway bridges with reduced congestion, delay, and accidents. To minimize intrusion in the transport flow, inspection and monitoring methods should be non?destructive, minimally invasive. They should be capable of yielding rapid and accurate inspection results allowing an adequate response from the asset manager. Research aims at including autonomously operating equipment (e.g. robotics), non?intrusive (remote or proximity) observation techniques, or other methods that ensure quality and performance control of the roadway bridges in time, more safely, more quickly and/or to a higher degree of accuracy and precision.The innovation subgroup in COST action TU1406 investigates novel condition monitoring and sensing technologies for the assessment of structural serviceability and safety. Advanced, integrated, cost-effective and reliable instrumentation solutions, techniques and concepts are looked at with the aim to provide data, that will be used to compute innovative performance indicators. In this context, this paper briefly reminds some significant challenges associated with bridge management and presents three examples of innovation in bridge monitoring and NDT investigation techniques