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
Adenoviruses have been widely developed as a vector for transgene delivery and protein expression. Early, intermediate and late genes are expressed in coordinated manner during the virus replicative cycle. The transition from early to late phases follows DNA replication and activation of the virus Major Late Promoter (MLP) to transcribe the major late transcription unit (MLTU), a primary transcript of ~28,000 nucleotides, which is alternatively spliced and polyadenylated to produce >20 mRNAs, encoding all but one of the viral structural proteins. Each late viral mRNAs share a common 5' noncoding sequence of 200 nucleotide known as the tripartite leader (TPL) sequence, consisting of leaders 1, 2 and 3, which mediates docking to the ribosome and translation via a cap-independent mechanism.
During the late phase of infection, host cell protein synthesis is suppressed, transport of cellular mRNAs from the nucleus to the cytoplasm is impaired, and transcription initiates predominantly from the virus MLP. Here, activation of the MLP coupled with amplification of viral genome results in up to 30% of cellular RNAs being MLP derived. Transcription of late viral 100K protein from the MLP induces inhibition of 5'cap binding complex, eIF4F, to represses translation of cellular mRNAs and switches the cell to cap-independent translation mechanisms for preferential translation of late viral proteins. Up to 90-95% of mRNAs translated are late viral transcripts containing the TPL. Cellular synthesis of RNA, DNA and protein are essentially hijacked to produce viral particles and, as a result, viral structural proteins transcribed from the MLP can comprise up to 40% of total cell protein.
Due to these favourable attributes, adenoviruses have been used extensively as expression platforms in large scale bioproduction of recombinant proteins and as helper systems in the production of adeno-associated virus (AAV) vectors. Whilst improving yields, one major issue with this approach is the risk of contaminating adenovirus particles in the final protein or AAV preparation. To overcome adenoviral particles contamination and improve the use of adenovirus as a vector for high-value recombinant protein and AAV vector production, we aim to develop a novel 'Tetracycline-enabled repressible adenovirus' (TERA) that exploits a Tetracycline-repressor system and the natural life-cycle of the adenovirus. A functional repressor binding site has not previously been inserted into the MLP in situ for the regulation of its expression in an adenovirus genome, primarily because the virus DNA polymerase coding sequence is in the opposing DNA strand. However, by strategic insertion of tetracycline repressor binding sites into specific loci of the MLP and encoding the tetracycline repressor under transcriptional control of the virus MLP, this regulatory element should enable doxycycline-dependent controlled expression of virus structural proteins.
In this approach, minimal cellular resources should be wasted as the transcriptional repressor of the MLP is directly linked to promoter activity. As the MLP transcribes the structural proteins of the virus, it also transcribes the repressor capable of repressing its own activity in the absence of any small molecule contaminants.
Hypothesis - an adenovirus encoding a self-repressing MLP should enable a negative feedback system for tight self-repression of adenoviral late structural proteins.
This approach should enable us to
I. Use adenovirus vector to retarget cellular resources for enhancing protein production by
A) maintaining viral genome replication to amplify transgene DNA within the cell
B) repressing viral MLTU so that the prevailing transgene mRNAs can efficiently be translated by cap-independent mechanisms
II. Use adenovirus vector for production of AAV viral vectors by
A) delivering adenovirus helper-functions
B) delivering and amplifying AAV DNA encoded in the E1-deleted region of the adenovirus
During the late phase of infection, host cell protein synthesis is suppressed, transport of cellular mRNAs from the nucleus to the cytoplasm is impaired, and transcription initiates predominantly from the virus MLP. Here, activation of the MLP coupled with amplification of viral genome results in up to 30% of cellular RNAs being MLP derived. Transcription of late viral 100K protein from the MLP induces inhibition of 5'cap binding complex, eIF4F, to represses translation of cellular mRNAs and switches the cell to cap-independent translation mechanisms for preferential translation of late viral proteins. Up to 90-95% of mRNAs translated are late viral transcripts containing the TPL. Cellular synthesis of RNA, DNA and protein are essentially hijacked to produce viral particles and, as a result, viral structural proteins transcribed from the MLP can comprise up to 40% of total cell protein.
Due to these favourable attributes, adenoviruses have been used extensively as expression platforms in large scale bioproduction of recombinant proteins and as helper systems in the production of adeno-associated virus (AAV) vectors. Whilst improving yields, one major issue with this approach is the risk of contaminating adenovirus particles in the final protein or AAV preparation. To overcome adenoviral particles contamination and improve the use of adenovirus as a vector for high-value recombinant protein and AAV vector production, we aim to develop a novel 'Tetracycline-enabled repressible adenovirus' (TERA) that exploits a Tetracycline-repressor system and the natural life-cycle of the adenovirus. A functional repressor binding site has not previously been inserted into the MLP in situ for the regulation of its expression in an adenovirus genome, primarily because the virus DNA polymerase coding sequence is in the opposing DNA strand. However, by strategic insertion of tetracycline repressor binding sites into specific loci of the MLP and encoding the tetracycline repressor under transcriptional control of the virus MLP, this regulatory element should enable doxycycline-dependent controlled expression of virus structural proteins.
In this approach, minimal cellular resources should be wasted as the transcriptional repressor of the MLP is directly linked to promoter activity. As the MLP transcribes the structural proteins of the virus, it also transcribes the repressor capable of repressing its own activity in the absence of any small molecule contaminants.
Hypothesis - an adenovirus encoding a self-repressing MLP should enable a negative feedback system for tight self-repression of adenoviral late structural proteins.
This approach should enable us to
I. Use adenovirus vector to retarget cellular resources for enhancing protein production by
A) maintaining viral genome replication to amplify transgene DNA within the cell
B) repressing viral MLTU so that the prevailing transgene mRNAs can efficiently be translated by cap-independent mechanisms
II. Use adenovirus vector for production of AAV viral vectors by
A) delivering adenovirus helper-functions
B) delivering and amplifying AAV DNA encoded in the E1-deleted region of the adenovirus
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| BB/P505031/1 | 01/10/2016 | 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 |