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

腺病毒作为转基因传递和蛋白表达的载体已得到广泛的发展。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是随着DNA复制和病毒主要晚期启动子（MLP）的激活来转录主要晚期转录单位（MLTU），这是一个约28,000个核苷酸的初级转录物，它被选择性地剪接和聚腺苷化以产生bbb20mrna，编码除一个外的所有病毒结构蛋白。每个晚期病毒mrna都有一个共同的5'非编码序列，长度为200个核苷酸，称为三联前导（TPL）序列，由前导1、2和3组成，通过帽独立机制介导与核糖体的对接和翻译。在感染的后期，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，转录主要从病毒MLP开始。在这里，MLP的激活加上病毒基因组的扩增导致高达30%的细胞rna是MLP衍生的。来自MLP的晚期病毒100K蛋白的转录诱导5'cap结合复合体eIF4F的抑制，从而抑制细胞mrna的翻译，并使细胞转向不依赖于cap的翻译机制，从而优先翻译晚期病毒蛋白。高达90-95%的mrna翻译为含有TPL的晚期病毒转录本。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细胞总蛋白的40%。由于这些有利的特性，腺病毒已被广泛用作重组蛋白大规模生物生产的表达平台和腺相关病毒（AAV）载体生产的辅助系统。在提高产量的同时，这种方法的一个主要问题是在最终的蛋白质或AAV制备中有污染腺病毒颗粒的风险。为了克服腺病毒颗粒污染，提高腺病毒作为高价值重组蛋白和AAV载体的使用，我们的目标是开发一种新的“四环素抑制腺病毒”（TERA），利用四环素抑制系统和腺病毒的自然生命周期。在腺病毒基因组中，由于病毒DNA聚合酶编码序列位于相反的DNA链上，因此在MLP中没有插入功能性阻遏物结合位点来原位调控其表达。然而，通过战略性地将四环素抑制因子结合位点插入MLP的特定位点，并在病毒MLP的转录控制下编码四环素抑制因子，该调控元件应该能够实现多西环素依赖的病毒结构蛋白的控制表达。在这种方法中，应该浪费最小的细胞资源，因为MLP的转录抑制因子与启动子活性直接相关。当MLP转录病毒的结构蛋白时，它也转录在没有任何小分子污染物的情况下能够抑制其自身活性的抑制因子。假设-一种腺病毒编码一种自我抑制的MLP，应该使一个负反馈系统能够对腺病毒晚期结构蛋白进行严格的自我抑制。这种方法将使我们能够：1 .利用腺病毒载体重新定位细胞资源，通过以下途径提高蛋白质产量：A)维持病毒基因组复制，在细胞内扩增转基因DNA； b)抑制病毒MLTU，从而使流行的转基因mrna能够通过帽独立机制有效地翻译。利用腺病毒载体通过a)传递腺病毒辅助功能B)传递和扩增腺病毒e1缺失区编码的AAV DNA来生产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