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The history of gene replacement therapies is a tale of scientific endeavor, persistence in the face of adversity, and world-changing discoveries. It encompasses breakthroughs in cell biology, molecular biology, biochemistry, structural biology, immunology, virology, oncology, engineering, and biotechnology. Yet despite the theoretical simplicity—overriding the disease-causing effect of a missing or faulty gene by inserting a working copy—there are still very few gene replacement therapies on the market today, 30 years since Rosenberg et al. demonstrated the potential of retroviral based gene transduction in humans (Rosenberg et al. N. Engl. J. Med. 1990; 323: 570–8).
Boom: The roaring nineties
The early 1990s were halcyon days for gene therapies. Researchers and clinicians alike believed that they held the key to curing all genetic diseases. Academics, investors, start-ups, and spinouts scrambled to enter this promising new market, driven by the hope of developing revolutionary treatments for gene-based disorders.
At this time, most gene therapy trials used adenoviruses to deliver the transgene into patients, a technique made possible by Professor Frank Graham’s work in the early 1970s to understand why some viruses are oncogenic, while others aren’t. In 1973, Graham—then a postdoc at the University of Leiden in the Netherlands—successfully created an adenovirus-transformed immortal human cell line, Human Embryonic Kidney (HEK)293 (Graham et al. J. Gen. Virol. 1977; 36(1): 59–74). HEK293 cells are easily transfected and contain the adenoviral E1 genes, which allow replication-incompetent adenoviruses to continue to grow in these cells. These characteristics make them an obvious choice for producing the large quantities of viral vector required for a human gene therapy. While gene therapy was booming, the human genome project, another remarkable feat of scientific investigation, was also underway. Fifteen years of global research collaboration resulted in the first publication of the complete human genome sequence in 2003.
Scientists now had access to not only the sequence of every human gene, but also maps detailing the location of these genes within chromosomes, and linkage maps to track the inheritance of genetic disease (Science Apr. 11, 2003 and Nature Apr. 24, 2003, full issues).
Success, stall, repeat (2003–2017)
With such a wealth of information available, it’s unsurprising that the gene therapy industry persisted in its attempts to revolutionize modern medicine.
In 2003, the China State Food and Drug Administration became the first health authority in the world to approve a gene therapy—an adenoviral vector carrying the P53 tumor suppressor gene—called Gendicine. However, we had to wait until 2017 before the U.S. Food and Drug Administration (FDA) approved the first gene therapy—Luxterna, for retinal dystrophy—for use in the United States (source: genetherapy.net).
Yet despite decades of research and investment, the scarcity of gene therapies currently on the market—and the cost of those that made it this far—speaks to the challenges still facing their development and manufacture. In particular, the challenge of cost-effective manufacture at the required speed, scale, and quality for clinical development remains to be overcome. For gene therapies like Luxterna, which target localised diseases and require only small doses, manufacture is relatively simple.
However, the publication of results from a successful hemophilia gene therapy trial in 2011 not only reinvigorated the gene therapy industry, but highlighted the need for new, scalable technologies to support the manufacture of gene therapies for systemic diseases that require high treatment doses (Nathwani et al. N. Engl. J. Med. 2011; 365: 2357–2365).
A look to the future: Transformative solutions to manufacturing challenges
At present, most gene therapy manufacturers rely on “scaling out” transient expression platforms. This is both costly and resource intensive, due to the large amounts of GMP-grade plasmid DNA required and/or the enormous cell culture footprint demanded by adherent cell cultures. But the future of gene therapy vector production undoubtedly lies in stable manufacturing solutions that can be easily “scaled up.”
At OXGENE™, our established expertise in DNA design and engineering, cell line development, upstream and downstream processing, and industry-leading automation is driving the transformation of our optimized AAV and lentiviral transient expression platforms toward alternative technologies for scalable, stable manufacturing.
We have strong foundations upon which to build. Our gene therapy production platforms are founded on proprietary SnapFast™ plasmid technology. These are modular plasmids, designed to work like “molecular Lego™,” using a catalogue of characterised DNA elements that can be easily and reliably inserted into specific locations within the plasmid. Our engineered AAV and lentiviral plasmids significantly improve packaging efficiency and viral titre, while our clonal HEK293 suspension cell line was specifically selected for optimal viral vector production (view data at: www.oxgene.com/News-and-Events/Gene-Therapy-Posters).
Joining forces with OXGENE in the early stages of gene therapy development allows our partners to establish and optimize transient production, including validated production up to 10-L scale, before transitioning to a stable technology platform for large-scale clinical manufacture. This provides the additional regulatory advantage of using the same genetic system throughout clinical development, as the stable platform retains the same expression cassettes and base cell line as the transient system.
Producer cell lines are an attractive alternative to transient transfection. Here, all the elements required for viral vector production, as well as the transgene of interest, are stably integrated into the cell’s genome. They therefore require no transfection and relatively little manipulation to scale up and consistently produce large quantities of viral vector, with lower batch-to-batch variation and at significantly lower cost. We’ve now successfully developed stable packaging and producer cell lines for lentivirus-based gene therapies.
We generated a stable lentiviral packaging cell line by transfecting packaging plasmids reconfigured with inducible vsv-g and gag-pol and constitutive rev expression into our HEK293 cell line. We then screened single-cell clones for growth kinetics, as well as stable—and inducible—expression of the viral genes. After several rounds of testing and analysis, we selected a single clonal lentiviral packaging cell line to expand, characterize, and optimize further. Process optimization has so far improved viral titer more than 10-fold.
The high level of optimization involved in perfecting OXGENE’s lentiviral packaging cell line makes this an excellent starting point from which to generate producer cell lines by stably transfecting a transfer plasmid containing a self-inactivating lentiviral genome and the transgene of interest. After another iteration of the cell line development process, the best-performing clones are expanded further and transferred for process optimization and scale-up to maximize viral titer.
With AAV, we’ve taken a different approach. We’re using our novel Tetracycline-Enabled Repressible Adenovirus (TERA) system as the basis for a stable AAV production platform. This uses an engineered helper adenovirus that contains a switchable negative feedback loop in the viral genome. This reduces helper adenovirus contamination to effectively zero and increases AAV yields. We have also shown that we can use this system to amplify both AAV rep and cap DNA from the cells’ chromosomes using the well established AAV Cis-Acting Replication Element (CARE). This technology allows the stable integration of DNA into cells, its subsequent amplification, and concomitant high protein expression levels, which provides a scalable, stable, and adenovirus-contaminant-free AAV manufacturing process.
Gene therapies are poised once again to transform the treatment of some of the world’s most devastating diseases. However, manufacturing challenges to date have hindered their development and approval. OXGENE’s ambition is to transform gene therapy manufacturing. By pioneering the development of tightly controlled and carefully optimized technologies to enable fully scalable, cost-effective, and high-quality gene therapy manufacture, OXGENE will help bring gene therapies to the patients who need them.
To learn more visit www.oxgene.com.