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

腺病毒已被广泛开发为用于转基因递送和蛋白质表达的载体。在病毒复制周期中，早期、中期和晚期基因以协调的方式表达。从早期到晚期的转变遵循DNA复制和病毒主要晚期启动子（MLP）的激活以转录主要晚期转录单位（MLTU），MLTU是约28，000个核苷酸的初级转录物，其被选择性剪接和聚腺苷酸化以产生>20个mRNA，编码除一种病毒结构蛋白外的所有蛋白。每种晚期病毒的mRNA都有一个共同的5'端非编码序列，称为三重前导序列（TPL），由前导序列1、2和3组成，通过帽非依赖性机制介导与核糖体的对接和翻译。在感染的晚期，宿主细胞蛋白质合成受到抑制，细胞mRNA从细胞核到细胞质的转运受到损害，并且转录主要从病毒MLP开始。在这里，MLP的激活与病毒基因组的扩增相结合，导致高达30%的细胞RNA是MLP衍生的。来自MLP的晚期病毒100 K蛋白的转录诱导5 '帽结合复合物eIF 4F的抑制，以抑制细胞mRNA的翻译，并将细胞切换到帽非依赖性翻译机制，以优先翻译晚期病毒蛋白。高达90-95%的翻译的mRNA是含有TPL的晚期病毒转录物。RNA、DNA和蛋白质的细胞合成基本上被劫持以产生病毒颗粒，因此，从MLP转录的病毒结构蛋白可占总细胞蛋白的高达40%。由于这些有利的属性，腺病毒已被广泛用作大规模生物生产重组蛋白的表达平台和生产腺相关病毒（AAV）载体的辅助系统。在提高产率的同时，这种方法的一个主要问题是在最终蛋白质或AAV制剂中污染腺病毒颗粒的风险。为了克服腺病毒颗粒污染并改善腺病毒作为高价值重组蛋白和AAV载体生产的载体的使用，我们的目标是开发一种新型的“四环素激活的可阻遏腺病毒”（TERA），其利用四环素阻遏系统和腺病毒的自然生命周期。功能性阻遏物结合位点以前没有原位插入MLP中以调节其在腺病毒基因组中的表达，主要是因为病毒DNA聚合酶编码序列在相对的DNA链中。然而，通过策略性地将四环素阻遏物结合位点插入到MLP的特定基因座中并在病毒MLP的转录控制下编码四环素阻遏物，该调节元件应该能够实现病毒结构蛋白的多西环素依赖性受控表达。在这种方法中，由于MLP的转录阻遏物与启动子活性直接相关，因此应浪费最少的细胞资源。当MLP转录病毒的结构蛋白时，它也转录能够在没有任何小分子污染物的情况下抑制其自身活性的阻遏物。假设-编码自阻遏MLP的腺病毒应该能够实现腺病毒晚期结构蛋白的紧密自阻遏的负反馈系统。这种方法应该使我们能够.使用腺病毒载体重新靶向细胞资源，通过A）维持病毒基因组复制以扩增细胞内的转基因DNA B）抑制病毒MLTU，使得主要的转基因mRNA可以通过帽非依赖性机制II有效地翻译，来增强蛋白质产生。通过A）递送腺病毒辅助功能B）递送和扩增腺病毒E1缺失区中编码的AAV DNA，使用腺病毒载体生产AAV病毒载体

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