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

腺病毒作为一种转基因载体和蛋白表达载体已被广泛开发。早期、中期和晚期基因在病毒复制周期中以协调的方式表达。在病毒主要晚期启动子(MLP)的DNA复制和激活之后，从早期到晚期的转变是转录主要的晚期转录单位(MLTU)，MLTU是一个由大约28,000个核苷酸组成的初级转录本，它被选择性地剪接和多聚腺苷化，产生&gt；20mRNAs，编码除一种病毒结构蛋白外的所有病毒结构蛋白。每个晚期病毒mRNAs共享一个由200个核苷酸组成的共同的5‘非编码序列，称为三部分前导(TPL)序列，由前导1、2和3组成，通过帽不依赖的机制介导与核糖体的对接和翻译。在感染后期，宿主细胞蛋白质合成受到抑制，细胞mRNAs从细胞核到细胞质的运输受到损害，转录主要从病毒MLP启动。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来自MLP。晚期病毒100K蛋白从MLP转录而来，诱导抑制5‘帽结合复合体eIF4F，以抑制细胞mRNAs的翻译，并将细胞切换到帽非依赖的翻译机制，以优先翻译晚期病毒蛋白。高达90%-95%翻译的mRNAs是含有TPL的晚期病毒转录本。细胞内RNA、DNA和蛋白质的合成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%。由于这些有利的属性，腺病毒已被广泛用作重组蛋白的大规模生物生产的表达平台和在腺相关病毒(AAV)载体生产中的辅助系统。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制剂中污染腺病毒颗粒的风险。为了克服腺病毒颗粒的污染，改进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‘四环素激活的可抑制型腺病毒’(TERA)，它利用四环素-阻遏系统和腺病毒的自然生命周期。以前还没有在MLP中原位插入功能抑制物结合位点来调节腺病毒基因组中的MLP表达，主要是因为病毒DNA聚合酶编码序列位于相反的DNA链上。然而，通过将四环素抑制物结合位点策略性地插入到MLP的特定位点，并在病毒MLP的转录控制下编码四环素抑制物，该调控元件应该能够依赖于多西环素来控制病毒结构蛋白的表达。在这种方法中，最少的细胞资源应该被浪费，因为MLP的转录抑制物直接与启动子活性有关。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自身活动的抑制子。假设-编码自我抑制的MLP的腺病毒应该能够对腺病毒晚期结构蛋白的紧密自我抑制产生负反馈系统。这种方法应该使我们能够I.使用腺病毒载体通过以下方式重定细胞资源以提高蛋白质产量：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，以便主要的转基因mRNAs可以通过帽不依赖的机制有效地翻译。用腺病毒载体生产AAV病毒载体通过)递送腺病毒辅助功能B)递送和扩增在腺病毒E1缺失区域编码的AAVDNA

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