AAVs are the most common vector for delivering in vivo gene therapies, but accommodating growing demand will require manufacturing innovation.
Gene Replacement vs. Gene Editing
Since its advent in the 90s, gene therapy has held promise as a way to defeat genetic diseases. Unlike CRISPR which edits the DNA, gene therapy keeps the existing DNA sequence in place, but replaces it with a healthy copy of the gene grown in vivo or ex vivo. For in vivo gene therapies, this sequence is typically delivered through an AAV (Adeno-Associated Virus) vector. Some of the common in vivo gene therapies include treatments for hemophilia, muscular distrophy, and cystic fibrosis.
In total, the FDA has now approved nearly twenty gene and cell therapy products, with an emphasis on blood and hemophilia-related disorders. But while further research can address lingering safety concerns with AAVs, new manufacturing processes will be needed to scale up production of these viral vectors in order meet growing demand.
Viruses Transport DNA, but Don’t Make Proteins
Before getting into how they’re made, it’s important to look at why viruses are used to deliver “replacement” genes. Viruses can carry DNA, but they cannot produce proteins from that DNA like a traditional cell can through transcription and translation. They’re great at transporting DNA, but need your cells to turn that DNA into a protein. In some ways, they’re like freight trucks that can bring raw materials into a factory, but lack the factory’s ability to transform those raw materials into a finished product.
Viruses engineered to carry repaired DNA for gene therapy can also be manipulated not to replicate beyond the target cells. This prevents the virus from sickening the patient. But it still means you can develop immunity to the viral vector, which is why gene therapy is often used as a one time treatment.
There are a few viral vectors that can be used to deliver gene therapy, but the AAV has become one of the most common because it can infect both dividing and non-dividing cells, and few people have been exposed to it so they don’t have immunity against it. Moreover, AAVs generally have low immunogenicity, that is to say they don’t produce a strong immune response from the body after insertion, which helps maintain their effectiveness as delivery mechanisms for new DNA.
There are some inherent challenges with AAVs, notably their DNA carrying payload is limited to 4,000 base pairs. Any sequence longer than that simply won’t fit. Longer strands can be accommodated by Adenoviral vectors, but those introduce an additional set of safety and quality challenges.
Are they Safe?
In addition to limited production volumes, AAVs and gene therapy still have safety concerns to overcome, particularly with high dosages. Two children died earlier this year in trials for a high dose AAV-delivered gene therapy for X-Linked Myotubular Myopathy (XLMTM). The particular capsid used for this trial, AAV8, has been used without any safety issues in 14 other trials, highlighting how sensitive AAV’s can be to shifts in their dosage and payloads.
Additional safety concerns have arisen around the potential to cause cancer observed in longitudinal studies of both dogs and humans. However, the data here is inconclusive. Nonetheless, the safety concerns obviously raise the risk for both gene therapy and its viral vectors, and for the time being have constrained their use in certain clinical trials.
Safety is the Top Priority, but Better Production Efficiency will be Essential to Meeting Future Demand
While additional research will be needed to address safety concerns, new manufacturing processes will be needed to ensure AAVs can be produced more efficiently and economically, which remains a challenge with existing manufacturing processes. This will be essential to meeting the growing demand for gene therapies.
Requiring substrates to which they can adhere, AAVs don’t scale up like mAbs (monoclonal antibodies) in a bioreactor. To grow they ultimately require a tremendous amount of plasmids, which both pushes up materials costs and creates potential quality risks. However, AAVs to date have faced limited pressure to scale up due to the low volumes and few approved gene therapies on the market. German drug developer Cevec claims it can deliver a cell line up front, similar to a master cell bank, from which AAVs can be drawn from, not unlike how mAb cell lines are stored. On top of that, they claim they can support standard 2000 L bioreactors, allowing a manufacturing scale not available with plasmid-intensive AAVs that struggle to sustain batches greater than 200 L.
Scaling Up Production
The main barrier to scaling up AAVs is putting their pieces together. There’s the “cap” or the process of building the capsid that encapsulates the virus, the “rep”, which allows the DNA to replicate, a helper virus needed to manage replication, and the payload, i.e. the nucleotides inside that actually deliver the therapy. By bundling it all together, Cevec believes they can overcome the volume limitations of existing approaches. But it remains unclear how they maintain quality and cost creating the cells. It would appear the upfront cost of doing this could still be significantly more expensive, made economical only through very high volumes that might be beyond current demand for existing gene therapies.
In contrast to Cevec’s approach, AGTC (Applied Genetics Technology Corporation) is proposing to accelerate scale up by using Herpes Simplex Viruses as “helpers” through a medium of BHK (Baby Hamster Kidney) cells. AGTC has been working on this approach for years, but like other AAV delivery methodologies, has been waiting for a gene therapy that could offer the volume to take advantage of it.
With just around twenty gene and cell therapy products approved by the FDA, AAVs primary use could still be in the clinic for another few years, allowing some time for more cost effective, high production growth techniques to develop. Either way, continued innovation in their manufacturing processes will be as important as innovation in the gene therapies they serve in order to serve a larger market.