Following pre-clinical and clinical success, adeno-associated viral vectors (AAVs), have become a leading tool for gene delivery in the treatment of a variety of human diseases. However, for patients to fully realize the benefits of these innovative therapies, the burden of cost will need to be addressed.

In this blog, Dr. Rajiv Vaidya, Head of Manufacturing Science & Technology (MS&T) at Andelyn Biosciences, explores the trends in AAV development and manufacturing, aiming to tackle this issue through improvements in scalability. As well as innovations in bioreactor design, optimization of upstream yields, and the generation of stable cell lines, Dr. Vaidya also examines how the changing regulatory environment necessitates  flexibility in gene therapy production.

 

AAV vectors for in vivo gene therapies

AAVs are small (20-25 nm in diameter), non-enveloped, non-integrating viruses that can deliver viral DNA to host cells in the form of an episome. Although their size signifies a relatively small cassette size (~4.7 kb), their mild immunogenicity and variety of serotypes mean they are quickly becoming the leading platform for in vivo delivery of new genetic material to patients’ cells. In part, their popularity in the gene therapy space can be attributed to advances in genetic engineering and molecular biology, which have helped to further enhance AAV capabilities. For example, the removal of viral coding sequences enables gene therapy developers to maximize AAV packaging capacity while lowering cytotoxicity and immunogenicity.

Following the first AAV gene therapy EMA approval in 2012, two more have followed, targeting ophthalmological and neurological diseases. Drug developers have taken notice of the success of these therapies, and as of 2022, there are more than 235 AAV gene therapies in the development pipeline at various stages.1

Although AAVs have shown potential for treating a broad range of indications, delivering these revolutionary gene therapies to patients is not without challenges, and noticeable trends are beginning to emerge as developers aim to overcome these hurdles.

 

1. A focus on scaling

One of the main barriers to effectively delivering AAV-based gene therapies to patients is the affordability of the drug product. This barrier is clearly demonstrated by the fact that, as of 2019, Zolgensma (Onasemnogene abeparvovec), an AAV-based gene therapy for spinal muscular atrophy, is the most expensive drug in the world, costing approximately $2.12 million (€1.9 million in Europe) per course of treatment.2 Considering the transient nature of the expression of genes introduced using AAV vectors, the possible need for subsequent rounds of treatment further adds to the pressure to relieve the cost of treatment.

AAV gene therapy developers are keen to reduce this cost burden to broaden patient access, and one fundamental way to achieve this is through increasing production scales. Gene therapy scalability has continued to improve since its advent, with advancements most noticeable in the capacity of bioreactors. Upstream processes that once relied on bioreactors with capacities around 200 L can now be conducted in volumes over ten times the size.

In tandem with scaling methods, AAV developers are looking at other ways to help improve the cost efficiency of the final product. Increasing yields and optimizing manufacturing technologies are key focuses, allowing more drug product to be produced per batch, thereby helping to decrease cost. By scaling with yield performance in mind and ensuring the yield profile is optimized early, viral vector developers can more easily achieve the productivity demands required for the whole product lifecycle. Efforts should be made to start downstream processes with a high virus titer and reduce product loss throughout purification. This process includes using media cultures and cell lines that can deliver higher yields and reagents that enhance transfection efficiency while safeguarding plasmid stability.

 

2. Upstream processes to meet scaling demands

Demands for scalability have most clearly manifested in AAV upstream processing, with growing trends centering around the production of stable cell lines and the generation of suspension cell culture systems.

Rather than relying on transient transfection, AAV developers are seeing a push for stable cell lines, where the genes for viral vector production are stably incorporated into the producer cell genome, allowing long-term expression. In comparison with transient transfection, the generation of stable cell lines means plasmid transfection methods do not have to be carried out for every batch, offering advantages in scaling. It can also help to decrease the cost of goods involved in the transfection steps – including GMP-grade plasmids and reagents – and can produce more homogeneous vectors of greater quality.

The desire for suspension cell lines for AAV production is also trending, as suspension platforms often offer advantages in scalability in comparison with adherent options. This is owing to the fact that adherent cell culture systems tend to require large amounts of floor space to accommodate 2D culture vessels at scale and copious manual handling.

Despite the industry seeing demand to move towards suspension and stable cell line options, their development can be arduous and there will be many technical challenges to overcome to see the benefits of these upstream options, requiring expertise and experience.

 

3. Regulatory changes impacting downstream processes

As well as responding to cost burdens, trends in AAV development and manufacturing are also driven by pressures applied by regulatory bodies. Compared to traditional biologics like monoclonal antibodies (mAbs), where regulations are well established and understood, the cell and gene therapy (C&GT) space is relatively new. Regulations are constantly evolving as our molecular understanding grows and technologies advance.

For AAV developers, this trend is currently in the spotlight – with regulatory changes causing a shift toward chromatography techniques in downstream processes. Previously, ultracentrifugation would have been considered the “gold standard” for purifying AAV, being an efficient method for removing empty capsids (capsids with no genetic material), which can impact the therapies’ efficacy and immunogenicity. As ultracentrifugation is considered an “open process,” there is an increased risk of microbial ingress, and regulators are pushing for alternative chromatography methods to be used instead to ensure patient safety.

The gene therapy space is still in relative infancy, meaning AAV developers can expect the need for flexibility to comply with changing regulations to continue for the foreseeable. By staying up to date with new innovations in the area through conferences, academic links, and investment in R&D, AAV developers and manufacturers can proactively weather the challenges of regulatory compliance.

Looking to the future

New, revolutionary gene therapies are entering the development pipeline every year, offering the potential to drastically change the lives of patients. As the gene therapy space grows, we can expect to see trends in AAV development and manufacturing being strongly driven by the desire to broaden access to these treatments by producing them at a larger scale, helping to lower production costs.

Although the adoption of innovative techniques and technologies will be needed to achieve these goals in a way that is compliant with continuously evolving regulations, appropriate implementation will rely on extensive expertise and experience in AAV production. Those thinking of making the journey to market should carefully consider finding support from a specialist contract development and manufacturing partner that could help to navigate potential changes and difficulties ahead.

To learn more about how Andelyn Biosciences can support your next AAV project, explore our gene therapy capabilities or get in touch today.

 

 

References

  1. https://www.delveinsight.com/blog/aav-vectors-in-gene-therapy-landscape
  2. Nuijten M. Pricing Zolgensma – the world’s most expensive drug. J Mark Access Health Policy. 2021 Dec 29;10(1):2022353. doi: 10.1080/20016689.2021.2022353. PMID: 34992762; PMCID: PMC8725676.