In its rather short life, the gene therapy field has been on a rollercoaster of experiences. While the initial hype was dampened by failures in clinical trials, the field is now experiencing a strong comeback. What was once seen as a hope, is now becoming a reality. But producing viral vectors, the essential delivery vehicles of gene therapies, remains challenging. CEVEC Pharmaceuticals has found a solution and developed a platform that can produce adeno-associated viral vectors (AAVs) as easily as if they were monoclonal antibodies.
As the gene therapy field grows, drug developers are confronted with the fact that most gene therapy products can’t be produced at the scale needed to meet growing demands. The reason: lack of adequate viral vector production technologies. What gene therapy developers desperately need is a production platform that can produce viral vectors simply, with no variations, avoiding cumbersome processes, and at reasonable costs.
I have spoken to Nicole Faust, CEO at CEVEC Pharmaceuticals about the challenges in viral vector production, how these are addressed with the company’s brand new production platform for AAVs, ELEVECTA®, and what it has in common with standardized production platforms that already exist for monoclonal antibodies.
Nicole, what are the current viral vector solutions in the gene therapy field? What are the advantages of AAVs over other vectors?
The great thing about gene therapy is that you can, in many cases, tackle the underlying cause of the disease. A lot of diseases today are just treated symptomatically, but with gene therapy, if the underlying cause is a gene defect, you can bring an intact copy of the gene into the patient or even repair the gene using genome editing tools like CRISPR-Cas.
To be able to do so, you need vehicles to deliver the gene. In most cases, although there are a number of non-viral approaches out there, the researchers use viral vectors. This makes a lot of sense because that’s what a virus does – it delivers genes to cells. We’re exploiting that feature of the virus, replacing the viral genes with the therapeutic gene, and using that viral vector to deliver the therapeutic gene to the target cells.
At the moment, there are three different viral vector types mainly used for gene-therapy approaches. One of them is the lentiviral vector, which has the advantage of integrating the gene into the cells, so it will stay there permanently. But lentiviral vectors also bear some risks because they can integrate into an unwanted position in the genome.
At the moment, lentiviral vectors are mainly used for ex-vivo therapies, in particular, because they are very good for transducing hematopoietic cells. Novartis’ Kymriah, for example, is a CAR-T therapy that uses a lentiviral vector to deliver the Chimeric Antigen Receptor (CAR) to T-cells outside of the patient. Then the modified cells are given back to the patient.
Second, there are adenoviral vectors, which were basically used when gene therapy started more than 20 years ago. They are still being used, but mainly for vaccination approaches. For example, there are some SARS-CoV-2 vaccines being developed at the moment with adenoviral vectors.
When we talk about in vivo gene therapies – actually delivering the therapeutic gene to the target cells inside the patient – then nearly always AAVs are used. The reason is that AAVs are non-pathogenic and the virus always needs the presence of a helper virus to replicate, and this makes it a lot safer than other viral vectors.
AAV is also a very interesting virus because it comes in a lot of different serotypes – different species of AAV. These serotypes correspond to distinctive structures on the surface of the virus, and that means that different tissues can be targeted. If you want to target neural tissue, for example, you’ll use a different AAV serotype than if you target the liver or muscles.
Another advantage of the AAV is that the particles themselves are very robust and very stable. They are easy to purify and once you’ve purified them, you can store them for a very long time without losing activity. All that makes them a nearly perfect tool for gene therapy.
What are the challenges of AAV production?
Upscaling is one of the biggest challenges in AAV production. The reason is that most of these therapies come out of universities, which means the first viral vectors were produced in a research lab by a method that would yield just enough vector material to do lab experiments. These methods work very well at this level, but they are not really scalable.
Also, in many cases, the viral vector production is based on adherently growing cells, so the cells need a substrate to adhere to in order to survive and divide. That means you can’t just use a huge 2000 L bioreactor, but you really have to provide the cells with a substrate and this is difficult at a large scale. Also, adherent cells are not a good solution for scalability because, very often, they still require animal-derived serum to grow, which presents a potential safety issue.
So instead, more and more suspension cell lines are being developed. But these cells still share one problem with adherent cells: Production of AAVs relies on a method called transient transfection.
What does that mean? To make an AAV, you have to bring into the cell different genetic elements. You need one plasmid that carries the rep and cap genes for the AAV life cycle and for producing the capsid; you need a second plasmid with the adenoviral helper gene; and a third plasmid with the therapeutic gene of interest, which is flanked by the recognition sequences that will allow the gene of interest to be packaged into the AAV vector.
So, you can imagine, transient transfection is convenient if you do it at a small scale in a lab, but it’s a challenge if you need to do it at several hundred liters. It’s not only a challenge with respect to the complexity of the process, but it also means you have to provide a lot of plasmid material.
The common understanding is that plasmids used for transient transfection in vector manufacturing for use in humans need to fulfill good manufacturing practice (GMP) requirements, which makes them very expensive. The plasmid costs can make up one-third of the production cost of a batch. That’s obviously a huge cost factor. You also need a transfection reagent. Often, there are sourcing issues and it can sometimes take up to half a year until you finally get the plasmid you need for your GMP production round.
How can these challenges be addressed?
At the moment, we’re still talking about ultra-rare diseases where batch sizes aren’t large. But a lot of common diseases, like Alzheimer’s and Parkinson’s, are currently in gene therapy trials. Once these trials are successful and they go into clinical phase III or even enter the market, then upscaling becomes a huge challenge. How will we produce sufficient amounts of the vectors in sufficient quantity and quality?
With ELEVECTA®, our new scalable, stable producer cell line technology for AAV gene therapy vectors, we wanted to address all the challenges I just mentioned. First of all, we have eliminated the lengthy and complex transfection step. Our platform does not require any transfection for the actual production of the AAV vector, which also means it doesn’t require any plasmid or transfection reagent. So, we don’t need any of the expensive raw materials.
ELEVECTA® is truly scalable because it’s basically made AAV production very similar to the well-established recombinant protein production methods. Using our platform, AAV production is very much like making a monoclonal antibody. With this, we’re addressing the major challenges that people are seeing for AAV production.
What is the science behind ELEVECTA®?
We’ve been thinking about how people have mastered the production of other biotherapeutics like monoclonal antibodies and why their production is scalable, and the answer is relatively simple: because they are using true, stable producer cell lines. That means all the genetic information that’s required to make the product is stably integrated into a producer cell.
Of course, we have to do cell line development in the beginning, but that only needs to be done once. Then, the producer cell line can be used for the production of the gene therapy vector for an unlimited period, as all the components are integrated into the cells’ genome.
I like making the comparison to monoclonal antibodies because making a monoclonal antibody used to be difficult, but it’s now become a common technology that can be outsourced to numerous service providers. We wanted to accomplish the same for AAVs. With our ELEVECTA® producer cells, we’re now able to do so.
Developing the ELEVECTA® technology was not trivial and we had to apply some tricks from the molecular biology toolbox, but we have been successful in the end. And we have very convincing data from true producer cells where we generated AAV vectors in large bioreactors with consistent productivity and quality.
One of the important quality measures is the full-versus-empty ratio because you always also generate empty particles that will not carry your gene of interest. That’s just inherent to AAVs. With our ELEVECTA® platform, we see a high ratio of full particles, which is what you are looking for, and it’s also consistent over different batches, making the process more robust and subsequent purification easier.
If a biotech company wants to access ELEVECTA® how can it best do that?
We want to make ELEVECTA® widely available and we want to see it in use for most of the future AAV gene therapy products. We will help interested parties make their specific producer cell lines with their specific gene of interest and their specific serotype
We are also supporting Pharma companies with a whole portfolio of products by offering a partner package and enabling them to do everything themselves, including cell line development.
But for most clients, we offer product-based projects. This means we generate stable producer cell lines as a service and then transfer those cells and the corresponding manufacturing processes to our clients who then use them under a technology license.
How do you see the landscape of gene therapy developing in the future and how is CEVEC planning to address these developments?
I believe that gene therapy has overcome the initial hurdles now and we will see many more products on the market in the future. But this also means that the therapy costs have to go down. At the moment, we’re talking about one million or two million for a treatment. That’s a big obstacle to making gene therapies commonly available. One important factor in addressing this issue is lowering production costs.
The standard size of a transient production run is about 200 L. With ELEVECTA®, it’s not a problem to scale up to 2000 L and beyond. So, in addition to reducing the material costs, our clients can benefit significantly from the economy of scale while using standardized processes, equipment, and facilities as known from antibody production.
Images via Shutterstock.com and CEVEC