Exploiting adenovirus mechanisms for the enhanced production of adeno-associated viral vectors and recombinant proteins

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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 cellB) repressing viral MLTU so that the prevailing transgene mRNAs can efficiently be translated by cap-independent mechanismsII. Use adenovirus vector for production of AAV viral vectors byA) delivering adenovirus helper-functions B) delivering and amplifying AAV DNA encoded in the E1-deleted region of the adenovirus