Advanced Technologies for Gene Therapy Products

Bespoke R&D solutions in virus-based biopharmaceuticals – from gene to bioprocess development

Gene Therapy at iBET

The development of advanced technologies for gene therapy products is a major research area at iBET for over 30 years. We work at the forefront of technology and knowledge to solve the most pressing issues in the field – improving productivity yields and process robustness as well as enhancing viral vector quality and its potency.

We focus on the development of platforms for production and purification of novel recombinant viruses or viral particles (e.g. based on retrovirus, lentivirus, adenovirus, and adeno-associated viruses – AAV). We employ a bespoke, integrated approach encompassing every stage from vector and cell line development to tech transfer to GMP production. Our interdisciplinary teams combine expertise and competences in molecular biology, cell biology, virology, and animal cell culture technology (from upstream to downstream processing and bioanalytics).

Expression Systems for Virus Manufacturing

We work on the optimization of recombinant viruses as well as their expression systems to improve both vector quality and manufacturing yields. Our strategies ultimately aim to enhance viral vector potency and clinical efficacy in delivering therapeutic gene(s) to target cells.

We employ advanced molecular biotechnology and synthetic biology tools, such as recombinase mediated cassette exchange (RMCE), inducible systems, CRISPR-Cas9 and other genetic engineering technologies, for the generation of modified human and insect cell lines. We also use mutagenesis, libraries and directed evolution to attain improved performance from the viral particles.

Bioprocess Development for Gene Therapy ATMPs

We develop integrated bioprocesses covering upstream and downstream processing technologies tailored to each viral vector type.

At the level of upstream bioprocessing, we devise customized and scalable GMP-compatible strategies where we establish and assess expression systems, cell culture systems and bioreactor type and operation mode, to monitor, control and optimize product titer and quality.

In downstream processing we develop solutions to increase product recovery yields, quality and safety while reducing manufacturing costs. Our cutting-edge purification processes are GMP-compatible, contributing to the design of disposable membrane cartridges and new chromatographic matrices, in collaboration with several industrial partners (e.g. Merck, Sartorius, Cytiva, Thermo LifeSciences).

Bioprocess Monitoring and Control

We apply “omics”-based tools including transcriptomics, metabolomics and fluxomics to guide cell genetic manipulation and media supplementation strategies to enhance desirable phenotypic traits and/or cell culture productivities. The in-depth characterization of producer cells and bioprocess key parameters translates into rational and target-oriented optimization strategies.

Product Characterization and Purity Assessment

We develop and implement a wide range of bioanalytical methods for detection of process-related impurities (e.g. chemical, host-cell derived, endotoxins, mycoplasma, etc), and in-depth characterization of viral vectors’ critical quality attributes (particle size, zeta potential, population distribution, and aggregation state). We employ chromatographic, electrophoretic and biochemical tools as well as cell-based assays, among other techniques. More info on our Bioproduction Unit page.

Related Technologies

Viral Expression systems

Design and construction of genetic cassettes for protein and virus expression (transient, viral mediated or stable expression in mammalian and insect cells) using advanced molecular biology and synthetic biology tools (e.g. inducible promoters, recombinase mediated cassette exchange - RMCE, CRISPR-Cas9).

Cell Line Development

Establishment of novel cell lines (mammalian and insect cell lines) or genetic enhancement of cell substrates through stable expression, knockout or knockdown, and clonal screening, clonality assurance.

Recombinant virus design and engineering

Design and genetic enhancement of recombinant viruses (lentivirus, retrovirus, adenovirus, adeno-associated virus, and baculovirus) and virus-like particles to improve the product therapeutic efficacy (specificity, tropism, transduction efficiency, immune response).

Upstream Processing

We employ our extensive know-how and expertise in cell culture systems for bioprocess development and intensification. Our state-of-the-art culture systems comprise glass, stainless steel and single-use stirred-tank bioreactors (up to 50 L) and single-use, rocking motion bioreactors (up to 25 L). Bioreactor operation modes used include batch, fed-batch, perfusion and continuous.

Downstream Processing

Design of integrated purification approaches for secreted and intracellular viral particles, which can be continuous or multi-column chromatographic purification systems tailored to the product features. Current approaches combine scalable membrane and chromatography-based technologies (e.g. ÄKTA Pilot, ultrafiltration systems) and filtration-based strategies (dead-end filtration and tangential flow filtration using hollow fibers and flat sheet cassettes).

Bioanalytical tools for virus research

We develop genetic tools and methods to detect, quantify and characterize viruses and viral particles (e.g. biosensors for virus detection and quantification). Other available technologies for characterization of viral particles include cell-based potency assays, capillary electrophoresis-based methods, nanoparticle tracking analysis, dynamic light scattering, mass photometry and surface plasmon resonance based-methods. Particles thermal and aggregation stability is characterized using different biophysical methodologies as (e.g. differential scanning fluorimetry).

Highlights

Expanding AAV vector cargo packaging using protein trans-splicing

Adeno-associated viral (AAV) vectors are one of the leading platforms for gene delivery. However, their small packaging capacity restricts their wider use for diseases requiring large-gene delivery. We have developed dual-AAV vector systems that rely on protein trans-splicing, based on split-inteins, to reconstitute large proteins.

Intein-containing genes can be split into two fragments and, subsequently, linked by a protein trans-splicing reaction, generating a full-length mature protein. We developed dual AAVs optimizing the trans-splicing reaction and the vector quality. We were able to achieve two-fold superior trans-splicing efficiencies than currently reported as the state of the art. Dual AAV in vitro co-transductions showed that 100% of cells carried successful protein reconstitution. By combining superior split-inteins with higher-quality vector preparations we could reduce vector doses 10-fold while maintaining high trans-splicing rates. (Ferreira et al 2023).

Scalable expression system for AAV vector manufacturing

In the scope of an industrial collaborative project, we established a manufacturing bioreaction process using HEK 293T cells in suspension culture adapted to serum free media.

AAV transient transfection production was optimized at small-scale (i.e. media screening, media supplementation, optimization of transfection parameters) and the optimal conditions were successfully validated in a 50 L single-use disposable bioreactor in collaboration with the Bioproduction Unit team. The project was concluded with the production of high-quality viruses (high ratio of full/empty particles) and the results disseminated in several oral and poster communications promoted by our industrial partner.

Understanding and monitoring of virus-based therapeutics

We established a new method based on capillary electrophoresis combined with laser-induced fluorescence to improve understanding and monitoring of the manufacturing process of virus-based therapeutics, mainly at the purification operation units.

Improving purification process understanding for viral vectors

New analytical technologies were implemented and integrated between unit operations aiming for deeper purification process understanding and faster decisions.

In this context, host cell DNA determination for downstream process of viral vectors and a new total virus particles quantification method are now available. Advanced analytical technology using fluorescence imaging and interferometry was implemented for the phenotypic characterization of extracellular vesicles and lentiviral vectors.

Identifying critical process parameters in scalable manufacturing of lentiviral vectors for gene therapy

Lentiviral vectors (LV) are the vector of choice in ex-vivo gene deliveries. To meet the increasing clinical demand, LV production platforms require improved productivity and scalability to enable cost-effective therapies.

In a study published by our team (Vaz et al, 2023), we established an improved serum-free suspension culture bioreaction process. Moreover, we identified key metabolic insights, unveiling important bioreaction parameters impacting vector yields. Moreover, we identified key metabolic insights, unveiling important bioreaction parameters impacting vector yields. After adapting HEK 293T cells to serum-free suspension cultures, LV yields were improved through transfection parameters optimization, process intensification and medium supplementation with nutrient boosters. Production at high cell densities increased volumetric titers up to 12-fold. Lipid supplementation was the most efficient metabolic optimization strategy further enhancing LV productivity by 3-fold. We also identified the cell concentration step, prior to transfection, as an important source of variability. Longer cell concentration processes, impaired cellular uptake of DNA polyplexes, impacting transfection efficiency and reducing LV titers down to 6-fold.

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Related Teams

Cell Line Development and Molecular Virology Lab

Ana Sofia Coroadinha

Head of Cell Line Development and Molecular Virology Lab

Development and engineering of virus-based biopharmaceuticals for gene therapy and vaccines.

Downstream Process Development Lab

Cristina Peixoto

Head of Downstream Process Development lab, Coordinator of Bioproduction Unit

Ground-breaking technologies for purification of new modalities

Cell-Based Vaccines Development Lab

António Roldão

Head of the Cell-based Vaccines Development Laboratory & Coordinator of Late-Stage R&D Unit

Multidisciplinary cutting-edge technologies to develop innovative bioprocesses for vaccine production

Advanced Cell Models Lab

Catarina Brito

Lab Head, Advanced Cell Models Laboratory

Leveraging ATMP development with advanced human cell models

Translational Immunology Lab

Nádia Duarte

Senior Scientist, Translational Immunology Lab

Strengthening immunology expertise at iBET, with a focus on developing tools and strategies to test and potentiate the effectiveness of advanced cell immunotherapies

Late-Stage R&D and Bioproduction Unit Team

António Roldão

Head of the Cell-based Vaccines Development Laboratory & Coordinator of Late-Stage R&D Unit

Late-stage R&D, scale-up and technology transfer for complex biopharmaceuticals production.

Highlighted Publications

Bioengineering for Cell-based Therapies

Reconstructed human pigmented skin/epidermis models achieve epidermal pigmentation through melanocore transfer

Pigment Cell & Melanoma Research

Michael J. Hall; Sara Lopes-Ventura; Matilde Neto; João Charneca; Patrícia Zoio; Miguel Seabra; Abel Oliva; Duarte Barral

2022