(Adeno-associated viruses) AAV Manufacturing has the potential to treat incurable diseases, however, their capsid protein-based shells can be heavy.
Hemophilia is an old genetic disease. This happens due to a deficiency in important blood clotting proteins. Even as recently as the 1960s, patients with severe hemophiliac disease were significantly less likely to live. The majority of them died prior to the age of 20.
Treatments that rely on the clotting protein and plasma are effective in extending a normal life expectancy. But these treatments are expensive and only temporary. Since one mutation in the two gene families causes hemophilia. There’s enormous interest and excitement to treat the condition and other genetic disorders with gene therapies that target blood cells. While the possibilities are amazing, gene therapies have to overcome numerous engineering, medical and logistical hurdles before they can be implemented effectively.
Adeno-associated viruses (AAVs) can be the most popular vector for delivery of the modified genetic material to the cells. The main benefit of AAVs is that it is non-pathogenic, meaning that it doesn’t cause illness. Another advantage, according to Professor Dr Jeffrey Hung of Charles River Laboratories one of the advantages is that “it’s extremely editable, meaning you can remove a large portion of the genome, and the virus will remain functional in carrying its DNA-based payment.”
New technological issues and AAV manufacturing challenges to overcome make it extremely durable. The protein found on the outside of the virus can fold itself into a functional form without gene therapy as a component. “It is like when a car could drive by itself without anyone inside,” explains Hung.
Therefore, as a result, a huge amount of them will not function with regard to the treatment involved when the therapeutic-laden viruses are made. “They are not able to fill some capsids. It’s not just black and white It’s a continuum” doctor. Glenn Pierce, Vice President of Medical and Global Blood Therapeutics for the World Federation of Hemophilia and director of two companies: Voyager Therapeutics and Global Blood Therapeutics. The number of capsids that are empty can be substantial, “as much as ten-fold more empty than those that are full.”
AAV Manufacturing Challenges: The Capsid Ratio
Production labs are working on these AAV manufacturing problems. “So how do we know that when we create an AAV-based gene therapy product, it is necessary to determine quantitatively what percentage of the product is made up of complete capsid, and how much is made up of empty capsid,” Hung says. Hung.
“The FDA cares deeply about the safety of clinical trials, therefore they pay close focus on the quality of batch-to-batch production. The empty-full ratio has to be constant.”
One of the method that, according to Hung one of the methods includes an analytic ultracentrifugation (AUC) instrument that measures the proportion of full capsids and empty capsids. Since the full and empty capsids have different masses as well as buoyancy levels, they could be separated using a centrifuge, stratified and separated in this procedure to increase the percentage that is full capsids.
While the FDA does not have special guidelines to follow, the requirements of patients are not as flexible and an empty capsid remains a virus that triggers an inborn immune response. Patients experience acute liver failure in several days” observes Pierce.
This response from the body is a problem that has been present for a long time in the field of gene regulation agents, but it’s not a one-time wave that will ride out. “Within three to four or five weeks, you will experience the adaptive immune reaction, which is T-cells that are cytotoxic,” says Pierce.
When you get AAVs like another virus, the protein is present on the surface of infected cells. The T-cells will eliminate those cells. This means that the dose is crucial -it is “if the dosage is too high, it can cause a massive reduction in cells,” Pierce says. Pierce since our body fights hard to fight the medication.
AAV manufacturing issues Cross-reactivity
However, even when they are successful, patients show a long-lasting antibody response. “We have seen from patients who were treated at the beginning of the 2000s that the response doesn’t diminish,” says Pierce.
In essence, you only get one shot at treatment since “either the way, you’ll get an enormous immunity response.” This is an issue for some patients since the various AAV variants are cross-reactive at 90-95% an immune reaction to one is an immune response that is universal to all.
Naturally, AAV infections can occur, and a child-related infection could shut down any treatment by AAV. However, despite the challenges with AAV manufacturing issues, says Pierce and the significant efforts to find alternatives, “no one has succeeded in achieving anything else.” The other technologies that are promising, like lentiviral vectors, are coming soon however they face unique manufacturing challenges that differ from those facing AAV methods.
Hung remains optimistic and shares an instance of motivation: “After one dose of injections, the animal started to run and walk…”Experts claim that there isn’t enough Clinical Manufacturing capacity to make the medication… which is why I’m thinking that this drug is truly a miracle. We have to bring this amazing treatment for patients.” Although the challenges of manufacturing AAVs for gene therapy can be quite daunting the industry is trying to overcome them.
AAV Gene Therapy Manufacturing
AAV Gene Therapy Manufacturing describes Adeno-associated virus’s properties, discusses their use as viral vectors, and explains manufacturing and control processes, including platforms, vector characterization and regulatory considerations.
Scientists harness the power of nature to maximize AAV production and ensure gene therapy safety.
Despite being the focus of the current pandemic, viral vices were not the main topic. However, for everyday scientists, viruses are an essential tool in their research toolbox.
Viruses are not vectors of disease. They are vectors of gene-edited transcripts and candidate genes as well as potential therapeutics. These tiny vessels aid in the scientific endeavour to understand and mitigate disease.
The key to gene therapy success is one such virus, the Adeno-associated virus (AAV). AAVs are tiny viruses with a lifespan of fewer than 5,000 kilobases. Scientists often equip AAVs using modified gene sequences and hire their services to deliver therapeutic genes to specific cells.
AAV is the preferred vector for gene therapy due to its low immunogenicity and safety profile. It can also infect many types of cells and tissues. However, it is not an easy task to manufacture AAVs.
Manufacturing Adeno-Associated Virus (AAV)
Three different plasmids are used to package AAV. One plasmid includes the gene of interest as well as a portion from the AAV genome. The second plasmid includes the AAV replication ( resp) as well as capsid ( ca_) genes. The tiny AAV cannot reproduce itself in nature. It requires help from an adenovirus, a second virus. The third plasmid includes key genes for AAV replication.
Although the system works, it is inefficient. Cells have a reputation for not being able to simultaneously take in multiple plasmids. Cells can take in one, two or all of the plasmids. Random integration results in increased production of empty viral capsids without any genomic information.
Only a few cells will be able to receive the perfect combination of plasmids. Scientists generate high viral titers in order to obtain optimal AAV infection for gene therapy. This increases the risk of adverse immune reactions.
AAVs and Adenoviruses can co-exist in nature and produce significantly higher viral levels with greater infectious potential. Wildtype adenovirus reproduces as many times as AAV.
This approach is not recommended by scientists and physicians. It can be efficient but it increases the chance of gene therapy contamination by adenovirus particles. What if wild-type adenovirus could help in AAV replication?
Two phases make up the life cycle for wild-type Adenovirus. The first phase contains non-structural proteins that are necessary for the replication of both AAV and adenovirus. The second phase produces structural proteins that are essential for wild-type Adenovirus assembly but not relevant to AAV replication.
OXGENE’s tetracycline-enabled self-silencing adenovirus vectors exploit the efficiency of wildtype adenovirus lives to produce amplified titers for highly infectious AAV. This prevents the second phase from occurring.