Regulation of gene expression in adeno-associated virus vectors in the brain

Regulated adeno-associated virus (AAV) vectors have broad utility in both experimental and applied gene therapy, and to date, several regulation systems have exhibited a capability to control gene expression from viral vectors over two orders of magnitude. The tetracycline responsive system has been the most used in AAV, although other regulation systems such as RU486- and rapamycin-responsive systems are reasonable options. AAV vectors influence how regulation systems function by several mechanisms, leading to increased background gene expression and restricted induction. Methods to reduce background expression continue to be explored and systems not yet tried in AAV may prove quite functional. Although regulated promoters are often assumed to exhibit ubiquitous expression, the tropism of different neuronal subtypes can be altered dramatically by changing promoters in recombinant AAV vectors. Differences in promoter-directed tropism have significant consequences for proper expression of gene products as well as the utility of dual vector regulation. Thus regulated vector systems must be carefully optimized for each application.     

Signaling to the chromatin during skeletal myogenesis: novel targets for pharmacological modulation of gene expression

Cellular differentiation entails an extensive reprogramming of the genome toward the expression of discrete subsets of genes, which establish the tissue-specific phenotype. This program is achieved by epigenetic marks of the chromatin at particular loci, and is regulated by environmental cues, such as soluble factors and cell-to-cell interactions. How the intracellular cascades convert the myriad of external stimuli into the nuclear information necessary to reprogram the genome toward specific responses is a question of biological and medical interest. The elucidation of the signaling converting cues from outside the cells into chromatin modifications at individual promoters holds the promise to unveil the targets for selective pharmacological interventions to modulate gene expression for therapeutic purposes. Enhancing muscle regeneration and preventing muscle breakdown are important goals in the therapy of muscular diseases, cancer-associated cachexia and aging-associated sarcopenia. We will summarize the recent progress of our knowledge of the regulation of gene expression by intracellular cascades elicited by external cues during skeletal myogenesis. And will illustrate the potential importance of targeting the chromatin signaling in regenerative medicine--e.g. to boost muscle regeneration.     

Regulated gene expression from adenovirus vectors: a systematic comparison of various inducible systems

Positively and tightly regulated gene expression is essential for gene function and gene therapy research. The currently-used inducible gene expression systems include tetracycline (Tet-on and T-REx), ecdysone, antiprogestin and dimerizer-based systems. Adenovirus (Ad) vectors play an important role in gene function and gene therapy research for their various advantages over other vector systems. Previously, we reported the inferiority of the Tet-on system as an inducible gene expression system in the context of Ad vectors in comparison with the Tet-off system. In this study, to identify an optimal system for regulated gene expression from Ad vectors, we made a rigorous direct comparison of these five inducible gene expression systems in three cell lines using the luciferase reporter gene. The highest sensitivity to the respective inducer was that of the dimerizer system, followed by the antiprogestin system. The lowest basal expression and the highest induction factor were both characteristic of the dimerizer system. Furthermore, the dimerizer and T-REx systems exhibited much higher induced expression levels than the other three systems. The elucidation of the characteristic features of each system should provide important information for widespread and feasible application of these systems. Overall, these results suggest the most appropriate inducible gene expression system in the context of Ad vectors to be the dimerizer system.     

Studying Gene Expression System Regulation at the Program Level

Understanding how gene expression systems influence biological outcomes is an important goal for diverse areas of research. Gene expression profiling allows for the simultaneous measurement of expression levels for thousands of genes and the opportunity to use this information to increase biological understanding. Yet, the best way to relate this immense amount of information to biological outcomes is far from clear. Here, a novel approach to gene expression systems research is presented that focuses on understanding gene expression systems at the level of gene expression program regulation. It is suggested that such an approach has important advantages over current techniques and may provide novel insights into how gene expression systems are regulated to shape biological outcomes such as the development of disease or response to treatment.     

米非司酮基因诱导调控系统

在基因表达和基因治疗研究中,需要目的基因在特定的时间以适当的水平实现其表达,目的基因过度表达或不适当的表达将影响实验结果,在疾病治疗中甚至会产生致命的副作用,实现对目的基因的表达时间和表达水平的精确调控是一个非常关键的问题。目前生物学家们已构建了多种新型的基因表达调控系统,其中米非司酮诱导调控系统具有诱导效率高,背景表达低,安全性高等诸多优点,是基因调控研究中的重要进展,也是目前最有应用前景的调控系统之一。本文就其结构设计和应用研究方面的进展作一简要综述。     

The future of human gene therapy

Human gene therapy (HGT) is defined as the transfer of nucleic acids (DNA) to somatic cells of a patient which results in a therapeutic effect, by either correcting genetic defects or by overexpressing proteins that are therapeutically useful. In the past, both the professional and the lay community had high (sometimes unreasonably high) expectations from HGT because of the early promise of treating or preventing diseases effectively and safely by this new technology. Although the theoretical advantages of HGT are undisputable, so far HGT has not delivered the promised results: convincing clinical efficacy could not be demonstrated yet in most of the trials conducted so far, while safety concerns were raised recently as the consequence of the "Gelsinger Case" in Philadelphia. This situation resulted from the by now well-recognized disparity between theory and practice. In other words, the existing technologies could not meet the practical needs of clinically successful HGT so far. However, over the past years, significant progress was made in various enabling technologies, in the molecular understanding of diseases and the manufacturing of vectors. HGT is a complex process, involving multiple steps in the human body (delivery to organs, tissue targeting, cellular trafficking, regulation of gene expression level and duration, biological activity of therapeutic protein, safety of the vector and gene product, to name just a few) most of which are not completely understood. The prerequisite of successful HGT include therapeutically suitable genes (with a proven role in pathophysiology of the disease), appropriate gene delivery systems (e.g., viral and non-viral vectors), proof of principle of efficacy and safety in appropriate preclinical models and suitable manufacturing and analytical processes to provide well-defined HGT products for clinical investigations. The most promising areas for gene therapy today are hemophilias, for monogenic diseases, and cardiovascular diseases (more specifically, therapeutic angiogenesis for myocardial ischemia and peripheral vascular disease, restenosis, stent stenosis and bypass graft failure) among multigenic diseases. This is based on the relative ease of access of blood vessels for HGT, and also because existing gene delivery technologies may be sufficient to achieve effective and safe therapeutic benefits for some of these indications (transient gene expression in some but not all affected cells is required to achieve a therapeutic effect at relatively low [safe] dose of vectors). For other diseases (including cancer) further developments in gene delivery vectors and gene expression systems will be required. It is important to note, that there will not be a "universal vector" and each clinical indication may require a specific set of technical hurdles to overcome. These will include modification of viral vectors (to reduce immunogenicity, change tropism and increase cloning capacity), engineering of non-viral vectors by mimicking the beneficial properties of viruses, cell-based gene delivery technologies, and development of innovative gene expression regulation systems. The technical advances together with the ever increasing knowledge and experience in the field will undoubtedly lead to the realization of the full potential of HGT in the future.     

Current status of transcriptional regulation systems

Many attempts have been undertaken to control transgene activity in mammalian cells. This is of importance for both applied biotechnology and basic research activities. State of the art regulatory systems use elements for transgene regulation which are unrelated to host regulatory networks and thus do not interfere with endogenous activities. Most of these regulation systems consist of transregulators and transregulator responding promoter elements that are derived from non mammalian origin. Apart from the tetracycline (Tet) regulated system which is most widely used for conditional gene expression at the moment, a number of new systems were created. These systems have been significantly refined and their performance makes them suitable for regulating transgenes not only in cellular systems but also in transgenic animals and for human therapeutic use.     

Muscle-mediated gene therapy

Transfer of therapeutic genes into muscle tissue holds promise for the treatment of a variety of muscular dystrophies, serum protein deficiencies and vascular proliferative disorders. Recent progress achieved in development of improved vectors allowed prolonged and efficient expression of genes encoding therapeutic proteins in muscle cells. The most important advances include: novel plasmid DNA vectors and methods for their efficient transfection in vivo, helper-dependent adenoviral vectors, allowing long-term gene expression and effective readministration in immunocompetent hosts and adeno-associated vectors. On the other hand, recent progress in this field has been facilitated by development of systems that enable regulated therapeutic gene expression in muscle tissue.     

仅基于RNA元件构建可诱导哺乳动物细胞基因表达的调控系统

大量的临床前和临床研究结果已表明基因治疗是理想的疾病治疗手段,然而如何实现治疗基因表达的精确调控仍然是研究人员面临的主要挑战。目前临床前研究常用的基因调控系统多基于控制转录,对反式转录激活因子和专门启动子元件的依赖限制了该系统的临床应用。最近,仅采用RNA元件构建的几种基因表达调控系统得到开发,其作用机制为核酶介导的RNA自我切割、RNA干扰、mRNA翻译启动或终止控制等。该类系统的调控活性由小分子配体反式控制,诱导基因表达的变化幅度可观,反应快速,在哺乳动物体内外均可实现。该系统结构模块化,调控活性可调节,可以克服现有转录调节系统的一些应用局限,对将来基因治疗的临床应用具有重要意义。     

EGR-1启动子在肿瘤基因治疗中的应用

EGR-1的启动子为早期生长反应因子-1基因上游约-550-0bp的一顺式作用元件。其活性受控于一些诱导剂,如电离辐射,联合EGR-1的启动子与治疗基因（如TNF-α、自杀基因）,用辐射能在肿瘤局部从时,空调控治疗基因的表达,使其产物局限于肿瘤局部,并发挥放疗的杀肿瘤效应,而放疗与转基因产物又有协同作用。放射治疗与基因治疗的配伍为肿瘤的治疗提供了新思路。     

Switching on the lights for gene therapy

Strategies for non-invasive and quantitative imaging of gene expression in vivo have been developed over the past decade. Non-invasive assessment of the dynamics of gene regulation is of interest for the detection of endogenous disease-specific biological alterations (e.g., signal transduction) and for monitoring the induction and regulation of therapeutic genes (e.g., gene therapy). To demonstrate that non-invasive imaging of regulated expression of any type of gene after in vivo transduction by versatile vectors is feasible, we generated regulatable herpes simplex virus type 1 (HSV-1) amplicon vectors carrying hormone (mifepristone) or antibiotic (tetracycline) regulated promoters driving the proportional co-expression of two marker genes. Regulated gene expression was monitored by fluorescence microscopy in culture and by positron emission tomography (PET) or bioluminescence (BLI) in vivo. The induction levels evaluated in glioma models varied depending on the dose of inductor. With fluorescence microscopy and BLI being the tools for assessing gene expression in culture and animal models, and with PET being the technology for possible application in humans, the generated vectors may serve to non-invasively monitor the dynamics of any gene of interest which is proportionally co-expressed with the respective imaging marker gene in research applications aiming towards translation into clinical application.