Manufacturing the Future of Accessible Gene Therapy

Gene therapies have the potential to change the lives of people with life-limiting, often incurable and rare diseases. The possibility to provide a cure is largely thanks to the use of gene delivery systems, commonly viral vectors – particularly adeno-associated viral (AAV) vectors and lentiviral (LV) vectors – to deliver a functioning copy of a gene to patient cells. These treatments could open the door to a one-dose, potentially curative treatment rather than a lifetime of medications. As the promise of gene therapies has grown, the number of clinical trials has expanded in recent years, with 372 clinical trials for gene therapies active in the first half of 2022. Within these trials, AAV vectors are the primary vector for gene delivery [1].

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This industry growth places challenges on Contract Development and Manufacturing Organizations (CDMOs) to manufacture enough AAV to support the increase in clinical trials in a timely and cost-effective way. In addition, as more gene therapies move through the development pipeline, the complex and costly processes associated with AAV manufacture may be reflected in the high costs of AAV-based gene therapies and inaccessibility of treatments to patients in need. As the industry develops, it is critical to ensure that any treatments that are currently early in the therapeutic pipeline can reach as many patients as possible after commercialisation.

Another present issue is the high treatment dose often required for AAV-based therapies, which raises safety concerns. Reducing the total volume of AAV vector administered to a patient, while maintaining the therapeutic potential, may improve the success of potential gene therapies [2].

To solve these problems, revolutionary manufacturing technologies are needed to massively improve AAV yield and scalability while reducing dose and improving safety. This article will highlight how one such technology may provide an answer.

Looking Beyond Plasmids

Plasmids have been a mainstay for viral vector production and continue to be an important technology for Good Manufacturing Practice (GMP) AAV manufacture. Advancements in plasmid engineering have also bolstered the success of manufacturing platforms, such as reconfiguration of the RepCap genes in AAV plasmids to increase yield as well as the removal of additional hexon genes in the adenoviral helper plasmid to enhance safety and performance.

There are, however, limitations of plasmid-based approaches which may only be overcome with truly disruptive technologies. Transient transfection of plasmids into HEK293 cell lines is inherently variable, with the possibility of inconsistent uptake and batch-to-batch variation and inconsistent scalability, limiting the viral vector yields that can be achieved using plasmid-based methods. Plasmids have the potential to be genetically unstable, which could become a substantial issue for maintaining quality and consistency when scaling up from a few copies to the billions required for treatment. There are supply chain bottlenecks in the manufacture of plasmids themselves, with relatively few facilities able to produce plasmids at a GMP grade, driving up costs and extending timelines. While plasmids will likely remain crucial tools for viral vector production, what does the next technological step look like for realizing the potential of gene therapies?

TESSA™: Plasmid-Free AAV Production

Manufacture of AAV using wildtype adenovirus is a highly efficient alternative to plasmid-based manufacture, but there are substantial contamination risks if adenovirus remains in the final product. The downstream processes then required to remove adenoviral contamination are expensive and time-consuming. TESSA™ technology offers a solution that many labs had worked for decades to try to provide. TESSA™ uses the adenoviral lifecycle to take a ‘back to nature’ approach for AAV manufacture while eliminating the risk of adenoviral contamination and the need for expensive decontamination.

TESSA™ makes use of the distinct early and late phases of the adenoviral lifecycle for AAV production. In nature, the early phase provides adenoviral help for high titre AAV manufacture but the late phase leads to adenoviral contamination. TESSA™ vectors use a doxycycline-inducible system for tight regulation of gene expression, switching on early genes enabling AAV production while tightly repressing late-phase genes and preventing adenoviral contamination.

AAV Rep and Cap genes and the gene or capsid of interest are stably encoded into two TESSA™ vectors which are used to infect HEK293 cells, providing a stable, scalable, plasmid-free production system with no need for transient plasmid transfection or separate adenoviral help (Figure 1). With the TESSA™ system, all of the cells’ resources are directed to producing AAV rather than adenovirus, so the yields are even higher than AAV manufacture using wildtype adenovirus.

Compared with triple transfection, TESSA™ technology has demonstrated a 30–40-fold increase in AAV titre when scaled up to 200L, demonstrating the benefits for AAV yield. Furthermore, the technology has the potential to be scaled up to 2000L. Because TESSA™ can generate more AAV than triple transfection from a set quantity of raw materials in a single bioreactor run, this could open the door to treating more patients in a more cost-effective way (Figure 2).

The issue with high AAV doses being required for therapeutic effect could be solved, at least in part, by improving particle infectivity. If particles can more easily infect target cells, this could reduce the total volume of AAV needed to treat a patient. Compared with triple transfection, TESSA™ technology has demonstrated an increased percentage of full capsids and improved infectivity. This suggests that TESSA™ could be helpful for reducing the total AAV dose needed to treat patients, and potentially improving the safety of gene therapies.

The Future of TESSA™

Following positive results reported in Nature Communications, in March 2022, WuXi Advanced Therapies launched TESSA™ for small-scale evaluation ahead of GMP manufacture [3,4]. Therapeutics companies and academic labs can now order a kit to evaluate TESSA™ in their own lab to assess suitability before embarking on their therapeutic journey [5]. Over the next few years, as more and more gene therapies enter the pipeline at early research stages, there is the hope that technologies like TESSA™ will streamline manufacturing processes and enable more patients to benefit from life-changing therapies.

References

[1] Alliance for Regenerative Medicine H2 2022 report, available here: Regenerative Medicine: The Pipeline Momentum Builds – Alliance for Regenerative Medicine (alliancerm.org)
[2] Kishimoto TK and Samulski RJ (2022). Addressing High Dose AAV Toxicity — ‘One and Done’ or ‘Slower and Lower’? Expert Opinion on Biological Therapy.
[3] Su W, Patrício MI et al (2022). Self-Attenuating Adenovirus Enables Production of Recombinant Adeno-Associated Virus for High Manufacturing Yield Without Contamination. Nature Communications.
[4] Launch of TESSA™ Technology – OXGENE
[5] https://oxgene.com/forms/order_tessa.aspx

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