Directed Evolution of Riboswitches in Mammalian Cells for External Control of Therapeutic Gene Expression

治疗性基因转移在癌症治疗中显示出很高的前景。复制缺陷和最近复制能力强的/溶瘤病毒被用作基因转移载体。后者结合了肿瘤细胞裂解和转移的治疗基因的放大。对治疗性基因表达的外部控制是可取的，甚至是必要的，以实现患者的时机和剂量，并作为一种安全措施，特别是对复制载体而言。我们提出了人工aptazyme，即配体依赖的自切割核酶，作为调节治疗性基因表达的创新工具。Aptazyme在本质上作用于RNA，独立于调节蛋白质-核酸的相互作用，是非免疫原性的，而且体积很小。这些是与广泛使用的诱导型启动子相比的关键优势，据报道，诱导型启动子在高拷贝数时也会失去调控，例如在病毒基因组扩增之后。使用以前未达到但仍不是最优的哺乳动物细胞诱导率的模型aptazyme，我们最近优化了aptazyme在哺乳动物转录单位中的定位。重要的是，我们已经证明，在复制缺陷和溶瘤腺病毒载体中，基因表达下调是完全有效的，与病毒复制和传播无关。该项目的目标是(I)通过定向进化，在哺乳动物细胞中鉴定具有高诱导率的开关适体酶，以及(Ii)利用这些开关来调节溶瘤腺病毒(OAD)对肿瘤靶向细胞毒配体的表达。对于定向进化，我们将通过随机化核酶和配体结合RNA结构域之间的连接序列来构建高多样性的aptazyme文库。然后，利用稳定转导的细胞，通过对端粒酶控制的GFP和自杀基因表达的顺序正向和负向选择，分别分离出诱导率高的Aptazyme。对不同配体作出反应的开关开关型和关闭型aptazyme都将通过在存在或不存在该配体的情况下进行选择来分离。由此产生的aptazyme将被测序，并在转基因研究中进行表征。接下来，我们将利用已鉴定的aptazyme来调节OADs对自然死亡配体TRAIL和免疫核糖核酸酶的表达。免疫核糖核酸酶显示抗体域靶向肿瘤表面分子的旁分泌毒性。我们最近证明了OADs可以表达功能性免疫核糖核酸酶，但需要对表达进行控制以减少对病毒复制的干扰。在这里，我们将在细胞培养和动物肿瘤模型中研究TRAIL和由OADS编码的EGFR特异性免疫核糖核酸酶在优化的转基因盒中的端粒酶控制和活性。最后，我们将分析肿瘤溶解和死亡配体的联合治疗。总之，我们将在实验室研究和生物医学中发现具有高基因调控潜力的核糖开关，并建立一种新的癌症治疗方案。

Therapeutic gene transfer shows high promise for the treatment of cancer. Replication-defective and more recently replication-competent/oncolytic viruses are used as gene transfer vectors. The latter combine tumor cell lysis with amplification of the transferred therapeutic gene. External control of therapeutic gene expression is desirable or even required to enable timing and dosing in patients, and as a safety measure, especially for replicating vectors. We propose artificial aptazymes, ligand-dependent self-cleaving ribozymes, as innovative tool for regulation of therapeutic gene expression. Aptazymes act RNA intrinsically, independent of regulatory protein-nucleic acid interactions, are non-immunogenic and of small size. These are key advantages compared to the widely used inducible promoters, which were also reported to lose regulation at high copy numbers, e.g. after virus genome amplification. Using a model aptazyme with previously unreached but still suboptimal induction rates in mammalian cells, we have recently optimized aptazyme positioning in mammalian transcription units. Importantly, we have shown that down-regulation of gene expression is fully functional in both replication-deficient and oncolytic adenovirus vectors, independent of virus replication and spread. The aims of the proposed project are (i) to identify by directed evolution ON- and OFF-switch aptazymes with high induction rates in mammalian cells and (ii) to exploit these switches for regulating the expression of tumor-targeted cytotoxic ligands by oncolytic adenoviruses (OAds). For directed evolution, we will generate high diversity aptazyme libraries by randomizing the linker sequence between ribozyme and ligand-binding RNA domain. Aptazymes with high induction rates will then be isolated by sequential positive and negative selection of aptazyme-controlled GFP and suicide gene expression, respectively, using stably transduced cells. Both ON- and OFF-switch aptazymes responsive to different ligands will be isolated by selection in presence or absence of the ligand. The resulting aptazymes will be sequenced and characterized in transfection studies. Next, we will exploit the identified aptazymes for regulating the expression of the natural death ligand TRAIL and ImmunoRNases by OAds. ImmunoRNAses show parakrine toxicity targeted by an antibody domain to desirable tumor surface molecules. We have recently demonstrated expression of functional immunoRNase by OAds, but expression control is required to reduce interference with virus replication. Here we will investigate in cell cultures and animal tumor models both aptazyme control and activity of TRAIL and EGFR-specific ImmunoRNase encoded by OAds in optimized transgene cassettes. Finally, we will analyze combined oncolysis and death ligand therapy. In conclusion, we will identify riboswitches with high potential for gene regulation in laboratory research and biomedicine and establish a novel regimen for cancer treatment.