Adenoviruses have been explored for gene therapy purposes for many decades. They are DNA viruses, with a double-stranded DNA genome of around 26-45 kilobases. Adenoviruses have been isolated from a broad range of species giving the adenovirus family an impressively wide host range. Adenoviruses typically cause mild infections which are self-limiting and as such, an adenovirus infection usually does not present with clinical symptoms. In addition, the vector can efficiently infect both resting and dividing cells and does not integrate into the host genome which avoids stable genetic modification.
The adenoviral replication cycle is tightly controlled by the expression of E1 proteins, providing an opportunity to generate replication deficient adenoviral vectors by deleting the E1 protein from the viral genome and producing so-called E1-deleted viruses in cell lines stably expressing the E1 protein. In addition, insertion of foreign DNA expressing one or more desired proteins under the control of heterologous promoters located in the former E1 region has been successful. As a consequence, high expression of desired proteins in target cells can now readily be achieved using E1-deleted vectors.
Adenoviral vectors are used in some of the most high-profile clinical trials in the field of gene therapy and have been approved as vaccine carriers to battle COVID-19 and Ebola. As with all vectors, their road to success has been paved with challenges.
Challenges in development adenoviruses as vectors
One of the first challenges in the development of adenoviruses as vectors was the rise of replication competent adenoviruses in product preparations. Here, E1 containing (and thus replication competent) vector particles were found to be present in product batches. This was considered a major safety issue as replication of recombinant adenoviral vectors in host cells had the potential to lead to tumor formation. The ultimate design of new packaging cell lines e.g. PER.C6 cells, void of any DNA sequence overlap between the vector and the E1 sequences present in the packaging cell line could successfully prevent the formation of replication competent particles in product preps.
Another challenge in the development of adenoviral vectors was the pre-existing immunity to many adenoviruses in humans. Here, owing to the widespread prevalence of wild-type adenoviruses in nature, as well as the vast numbers of serotypes circulating in humans, there is a substantial chance that the host will have encountered the adenovirus previously and therefore possesses pre-existing immunity against the virus. In such instances, and depending on the route of administration, the host immune system may clear the vector product before it has had a chance to infect target cells and express the desired protein(s) thus severely limiting product efficacy. To circumvent pre-existing immunity, researchers turned to rare human serotypes and serotypes from non-human origin. To date, there are many different adenoviral vectors available to circumvent pre-existing immunity. Available vectors are either selected from rare human serotypes, non-human primates or alternative species like dogs or goats.
Successfully avoiding host pre-existing immunity has helped to re-position adenoviral vectors as important tools in future gene editing and vaccine product development. Here the human Ad26 vector from Janssen Vaccines and the Chimpanzee derived vector from Oxford have taken dominant roles in building safe and effective vaccines against SARS-CoV-2, the cause of COVID-19 disease. Mass vaccination campaigns using these vectors have now revealed another challenge and that is the induction of severe clotting in humans at extreme low frequency. Around the globe researchers are currently working to understand the basic scientific principles of this rare phenomenon. It can be expected that a thorough understanding of the underlying pathway(s) of adenoviral vector mediated clotting will be elucidated soon, most likely resulting in further modifications to the adenoviral vector backbone.
Current uses of adenoviral vectors
The adenoviral vector represents one of the most studied vector systems currently available to researchers and product developers. The wealth of knowledge and available databases on vector development, manufacturing, pre-clinical data and clinical data is unprecedented. The thorough R&D scrutiny of adenoviral vectors have provided tools and methods for assessing both safety and efficacy prior to conducting clinical trials and have proven extremely robust. This is one of the biggest strengths of the adenoviral vector platform. In addition, the adenoviral vector system represents by far the most mature vector platform when it comes to manufacturing. This is mainly due to the fact that several vectors have been approved as products on the market. Vector production trains, purification methods and testing/release protocols have been thoroughly scrutinized and completely streamlined. The latter has resulted in a thorough understanding of the volumetric output of the vector platform in relation to the cost of goods (COG) of manufacturing. Such analyses have shown that the adenoviral vector platform is extremely competitive on pricing compared to any other vector platform currently being researched.
Many of the factors outlined above have significantly contributed to the rapid response by both AstraZeneca and Janssen in the deployment of their adenoviral vector based COVID vaccines for emergency use. Here, plug-and-play vector platforms and a well-known and established regulatory path have been crucial for rapid deployment.
This is why the adenoviral vector platform is as sought after as ever. Indeed, adenoviral vectors are the vector of choice for many different vaccine strategies including HIV, Zika, malaria and tuberculosis to name but a few. In all of these programs currently being pursued, pre-clinical studies have demonstrated effective protection against disease. In addition, these studies have proven that adenoviral vector-based vaccines provide long term and durable protection, avoiding the need for repeat vaccination. The latter is crucial in the logistics of vaccine deployment especially in developing countries. There is no doubt that many new vaccines using the adenoviral vector platform will make it to market.
When it comes to gene therapies, adenoviral vectors have been very successful where localization of the treatment is required. The ability to modify the fiber protein and inject the adeno vector locally means that genetic therapies can be closely confined to specific regions of the body. Genetic therapies for treating the eyes as well as targeting specific tumors have been well established using adenoviral vectors. Another popular use is in ex-vivo therapies where for instance veins are removed and treated with adeno vectors before being returned to the body (vein grafting). Here, adenoviral vector therapies have a highly promising preclinical success rate and have demonstrated the ability of local gene transfer confined to the tissue of interest.
Adenovirus vector manufacturing
Despite the broad tissue tropism of the adenovirus family, the vast majority of them grow on an engineered cell line called HEK293. This is a commercially available cell line that can be used in the research lab as well as the production hall. While there are different cell lines that can be used to produce different adenovirus serotypes, the fact that most will grow in HEK293 offers a great advantage. The prevalence of HEK293 cells in the development of adenoviral vectors means that from a regulatory standpoint, they are much less risky than novel cell lines with no track record.
Adenoviral vectors are also amenable to genetic modification. It is common and relatively straight forward to change the fiber proteins on the surface of an adenovirus. Such a change can have significant effects on the tissue tropism, helping to guide the virus to specific tissues in order to deliver its payload. This is a very useful feature for treatment and can also help determine the administration route. This provides the adenoviral vector platform with high flexibility when it comes to therapeutic options. This feature is especially useful for developing oncolytic therapy where an adeno vector can be engineered to specifically target tumor cells or even specifically replicate in tumor cells.
In conclusion, the impressive track record of adenoviral vectors coupled to the platform flexibility and versatility in developing new vectors make them an important and extremely relevant tool for developing therapies and vaccines.
