Exploiting adenovirus mechanisms for the enhanced production of adeno-associated viral vectors and recombinant proteins
Lead Research Organisation:
UNIVERSITY OF OXFORD
Department Name: Oncology
Abstract
Abstracts are not currently available in GtR for all funded research. This is normally because the abstract was not required at the time of proposal submission, but may be because it included sensitive information such as personal details.
People |
ORCID iD |
| Leonard Seymour (Primary Supervisor) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/P505031/1 | 30/09/2016 | 24/01/2021 | |||
| 1865453 | Studentship | BB/P505031/1 | 01/02/2017 | 31/01/2021 |
| Description | Adenoviruses were first discovered in the 1950s and have been since been studied extensively as a model system for understanding the fundamental cellular process on how genes, that are coded within our DNA, are transcribed and translated into proteins. Additionally, human adenovirus, particularly serotype 5 (Ad5), has received tremendous attention for use in large scale bioproduction of recombinant proteins and as a 'helper' virus for the production of adeno-associated virus (AAV) vectors, currently the vector of choice for gene therapy. While high yields of recombinant proteins and AAV vectors can be produced using adenoviruses, one major issue is the risk of contaminating adenovirus particles in the final product limiting its widespread application. A key novel finding in our research so far involves a breakthrough in genetic modification of the adenovirus - in creating a self-repressing adenovirus, wherein the replicative life-cycle of the virus is efficiently truncated, while other advantageous biological properties that enables, such a DNA replication within the nucleus of the cell, is maintained. Specifically, the adenovirus life-cycle is regulated in a timely and coordinated manner wherein the transition from the early to late infection phase follows activation of the virus major late promoter to produce the viral structural proteins that form the capsid particle. In our work, we were able to engineer the Major late promoter, within the adenovirus, to allow repression of its activity when bound by a repressor protein. We have also engineered the repressor protein to be produced under the direct control of the major late promoter, effectively creating a negative feedback loop to inhibit replication of the adenovirus. Following infection into a complementing cell, this engineered adenovirus is able to proceed through the early phase of its life-cycle, but as its transitions to the late infection phase, activation of the viral major late promoter results in the production of the repressor protein to inhibit the production of its particle structure and hence infectious adenoviruses. Historically, this has never been achieved due to the in situ location of the major late promoter within the genome of the adenovirus. Currently, this adenoviral vector is being explored for the production of recombinant proteins and results have been particularly positive. Significant increases in the production of recombinant antibodies have been achieved that are free of contaminating adenoviruses. To extend the use of this self-repressor adenovirus vector for the scalable production of AAV vectors, currently a major limitation for its use in gene therapy, we have further engineered our novel adenoviral vectors to encode all the genes and component required for AAV production. This novel AAV production system simply requires the co-infection of self-repressing adenovirus vectors (encoding all of the AAV genes and component required in the process) into mammalian produce cells to produce large amounts of AAV vectors that are free of contaminating adenoviruses. This new AAV production system have considerable advantages, such as scalability, increase yield and quality of AAV particles, over the traditional method of AAV production, which involve transfection of plasmid DNA encoding the AAV genes into producer cells. Attempts to encode AAV DNA into adenovirus vectors for producing AAV vectors have been extensively explored in the past. However, results have been poor because AAV genes and its DNA coding sequences are highly cytotoxic to adenovirus replication. Here, we have taken an approach guided by rational design to alleviate the toxicity induced by the AAV genes, to enable each of these components to be stably encoded within our self-repressing adenovirus vectors. |
| Exploitation Route | Currently, we are exploring the use of our novel adenovirus for the contaminant-free and economical production of viral vectors (AAV) for gene therapy and biomolecules such as antibodies. However, our findings are also useful in understanding the biology of the adenovirus, as the effect of uncoupling the DNA replication phase of the virus from encapsulation and particle generation could have unforeseen effects on the virus and how it interacts with cellular factors. Furthermore, selectively turning-off the ability of the adenovirus to create structural particles should enhance the safety profile of its use as an oncolytic virus or gene therapy vector. |
| Sectors | Manufacturing including Industrial Biotechology Pharmaceuticals and Medical Biotechnology |