Adenovirus (Ad) vectors are used to develop gene therapy products and vaccines. Decades of research stand behind these vectors which have gone through several iterations and improvements to make them what they are now. As mentioned in a previous article, the E1 proteins which control the adenoviral replication cycle were removed in very early iterations of the adenovirus vectors. This prevented the virus from replicating in humans, thus only allowing the virus to deliver its genetic payload. In addition, the E3 region, which is not essential for viral propagation, was removed. The space freed up in the wildtype virus genome by the removal of E1 and E3 allowed for the insertion of up to 6.5 kb of foreign DNA.
This was the first generation of Ad vectors. In the second generation more improvements were made when E2a, E2b and E4 were removed. The removal freed up yet more space in the genome for up to 10.5 kb of transgene DNA to be inserted into the chromosome. This was particularly relevant for researchers desiring to put multiple antigens into one vector. For instance, in pursuit of AIDS vaccines it was shown that a combination of HIV virus-derived envelope together with Gag and Pol provided broad immune responses. The deletion of more genes encoding for E proteins from the viral backbone also improved the safety of Ad vectors by making it less likely that spontaneous recombination events during the vector propagations would lead to replication-competent viral particles. The deletion of multiple E-encoding genes from the viral genome also led to a significant reduction in viral gene expression in host cells, lowering a cytotoxic T-lymphocyte response against the vector itself. This made second generation Ad vectors much less likely to be cleared by the immune system.
Third generation Ad vectors are void of all viral sequences except for the inverted terminal repeats, which are the signals for the DNA to be effectively packaged into the adenoviral capsid proteins. These high-capacity adenovirus vectors can package up to 36 kb of foreign genetic material. To produce these vectors, adenoviral helper viruses are required to produce the proteins needed for replication and packaging of the Ad vector genome.
Recombinant Ad vectors have been around for a long time and the active research around them has led to great leaps forward in their usage and in the fields of gene therapy and vaccine production. Currently, it is possible to make conditionally replicating Ad vectors, which only replicate inside tumor cells. When this is combined with the effective cell targeting provided by transgenic knob proteins on the capsid surface, Ad vectors become a truly powerful tool for cancer therapies as well.
Main advantages for Adenovirus vectors
Ad vectors have advanced further than any other vector system owing to continued active research from academia and industry together. This research has made Ad vectors more effective while also making them safe. Modern Ad vectors have four key advantages in gene therapy and vaccine development.
- High transduction efficiency in dividing and quiescent cells
This is one of the reasons that adenoviruses were first considered as vectors for gene therapy and vaccines. Ad vectors can deliver genetic cargo to cells very efficiently, so that therapeutic levels can be achieved with fewer viral particles. This is extremely important for in vivo systemic applications where high concentrations of Ad vector are typically more challenging.
- Epichromosomal persistence in cells
This feature of Ad vector has led to some very specific and highly sought-after applications. Persistence of the vector is important to allow the genetic payload to be delivered, transcribed, and expressed as therapeutic proteins. Without the ability to persist, these therapies would not be present long enough to be effective. However, a common mode of persistence for viruses is chromosomal integration. This is often undesirable or unnecessary in gene therapy and carries many safety concerns specifically for DNA vectors. Because Ad vectors do not integrate into the chromosome of the host cells, there is no risk that they will permanently alter the host genetic make-up.
- Broad tissue tropism
A wide variety of wild-type adenoviruses have been modified to create Ad vectors, providing a range of different tissue tropisms. More specific tropism has been developed in many therapeutic cases thanks to the genetic manipulation of the knob protein, which is the protruding end of the fiber capsid that is primarily responsible for host cell attachment. This protein is the key to unlock the entry into specific cells through receptor-specific binding. By modifying the knob protein, it is possible to make Ad vectors that can be precisely targeted to specific tissues.
- Scalable production
Gene therapy products that cannot be scaled to commercial manufacture cannot be used to treat real-world diseases. As we saw during the rollout of the COVID-19 vaccines, Ad vector-based products can be rapidly scaled to meet market demand. To meet demand for the COVID-19 vaccine, each 5-day production cycle readily delivered over 15 million doses of vaccine to battle SARS-CoV2 virus.
Together, the described benefits of Ad vectors provide a broad and versatile platform to achieve sought-after clinical breakthroughs in the treatment and prevention of debilitating and life-threatening diseases
Clinical applications of Ad vectors can be broadly split into two categories: vaccines and gene therapies.
Basic research in pursuit of improved Ad vectors revealed that the high prevalence of wild-type adenoviruses in the human population has led to widespread pre-existing immunity, particularly to some human serotypes like Adenovirus serotype 5. Developments in the field have turned this potential drawback into an important feature of Ad vector-based vaccines.
With respect to vaccine development, the main purpose of a DNA-based vector is to deliver epitopes from other viruses to host cells and to ensure production of such epitopes in order to raise an broad and effective immune response against the virus. Over the iterations of development, Ad vectors have become better at delivering increasingly large nucleic acid payloads. In addition, the immunogenicity of some Ad vectors has been tweaked and harnessed to boost the production of pro-inflammatory cytokines that can enhance the humoral and cellular immune responses.
A successful application of this approach is the Ad vector-based Ebola vaccine developed by Janssen. This vaccine, which takes advantage of Janssen’s rare Ad26 serotype, induces specific antibody and T-cell responses against Ebola virus. Clinical results demonstrated that the vaccine elicits a powerful humoral immune response in humans that persists for more than a year. Rapid and durable T-cell responses using Ad26 were also seen in adenovector-based COVID-19 vaccines, which induced strong humoral and cellular immune responses in 100% of clinical trial participants after two doses.
Ad vectors have also been widely used for the production of cancer vaccines. Current vaccine research is focused on prostate cancer, human papilloma virus, colorectal, and pancreatic cancers. In addition to cancer prevention through vaccines, Ad vectors are also used for anticancer therapies.
Anticancer therapies using adenovirus vectors fall broadly into three main categories:
- Delivery of suicide genes
Many tumor cell types proliferate rapidly and have a dysfunctional p53 tumor suppressor pathway. Ad vectors can be engineered to induce p53 expression inside tumors, triggering cell death. Another successful application has been using Ad vectors to deliver genes that convert a pro-drug into an active cytotoxic agent. For example, the enzyme purine nucleoside phosphorylase converts the pro-drug fludarabine monophosphate into fluoroadenine, which kills proliferating cells. Trials using this enzyme have already been conducted using Ad vectors. The ability of Ad vectors to remain localized is extremely valuable when delivering cytotoxic therapies that can harm heathy cells if the treatment is not constrained to the target cell populations.
- Delivery of immune-regulatory genes
Ad vectors can also be loaded with genes that stimulate an antitumor immune response. Antitumor interferon-β and interferon-α-2b have both been safely delivered to the lungs of patients via intrapleural injection.
- Chimeric and tropism-modified oncolytic Ad vectors
A problem that often arises during cancer therapies is poor recognition of tumors by immune cells and Ad vectors. In this case, Ad vector knob proteins can be modified to bind more strongly to receptors on the surface of the tumor. One example of this is in an Ad vector used to treat ovarian cancer where the entire fiber knob domain of Ad5 was replaced with that of Ad3. The result was targeted ablation of ovarian cancer cells displaying elevated levels of Ad3 receptors.
Adenoviral vectors have come a long way since their initial use several decades ago. Research has shown that they can be used extremely effectively for a number of gene therapy and vaccine applications.
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