Targeting HIV-1 gene expression: towards a new approach for an antiretroviral therapy

在HIV - 1复制过程中，病毒（+）RNA基因组逆转录，转化为双链DNA并整合到宿主细胞基因组中。原病毒的转录产生多顺反子前mrna，其以剪接或未剪接的形式离开细胞核。很大一部分剪接的mrna也含有内含子。由于这种病毒严重依赖于这些剪接和未剪接mrna的平衡表达，因此这种比例的任何干扰都会显著影响HIV - 1的感染性和发病机制。最近，我们可以证明HIV-1基因组中的两个保守剪接调控元件，GI3-2和ESEtat元件在病毒mRNA物种（如vif， vpr和其他mRNA）的产生中起主要作用。此外，这些元素的突变抑制了病毒的复制。关于一种针对HIV-1基因表达的替代性抗逆转录病毒疗法的开发，我们用反义寡核苷酸掩盖这些剪接调节元件，也导致HIV-1颗粒产生的阻断。作为寡核苷酸，我们使用锁定的核酸（LNAs），由于额外的亚甲基桥，修饰的核苷酸被限制在理想的沃森-克里克结合构象中。除了与其他反义寡核苷酸相比的许多优点外，LNAs可以在没有转染试剂（裸体）的情况下被递送到细胞中，这使得这些寡核苷酸具有治疗应用的吸引力。我们提出LNAs为开发针对HIV-1基因表达的新型抗逆转录病毒疗法提供了一个有前途的工具。我们的初步结果表明，裸体传递的LNAs，甚至比转染的LNAs，对病毒复制的影响非常大。令人惊讶的是，LNAs在没有辅助的情况下传递，在细胞质中定位，并在这里诱导含有其目标序列的病毒mrna的降解。如果LNAs在较晚的时间点也在细胞核内积累，并且通过屏蔽剪接调节蛋白的结合位点而干扰病毒的选择性剪接，则必须进行分析。因此，我们希望在一个时间过程实验中分析体操传递的RNA的细胞定位和分布，并揭示潜在的RNA介导的RNA降解机制（靶效应）。此外，我们希望研究可能的脱靶效应，包括先天免疫反应的刺激。此外，我们的目标是在HIV-1基因组中确定额外的LNA靶点，以开发LNA鸡尾酒作为替代抗逆转录病毒疗法。

During HIV 1 replication the viral (+)RNA genome is reverse transcribed, converted into double stranded DNA and integrated into the host cell genome. Transcription of the provirus generates a polycistronic pre-mRNA that leaves the cell nucleus either in a spliced or unspliced form. A significant fraction of the spliced mRNAs also contains introns. Since the virus critically depends on a balanced expression of these spliced and unspliced mRNAs, any disturbance of this ratio dramatically affects HIV 1 infectivity and pathogenesis. Most recently, we could show that two conserved splicing regulatory elements within the HIV-1 genome, both the GI3-2 and the ESEtat element play a major role in the generation of viral mRNA species such as vif, vpr and tat mRNAs. Furthermore, mutations of these elements inhibited viral replication. With regard to the development of an alternative antiretroviral therapy, which targets HIV-1 gene expression, we masked these splicing regulatory elements with antisense oligonucleotides also resulting in a block of HIV-1 particle production. As oligonucleotides we used locked nucleic acids (LNAs), modified nucleotides which are constrained in the ideal conformation for Watson-Crick binding due to an extra methylene bridge. Besides many advantages over other antisense oligonucleotides LNAs can be delivered into cells without transfection reagents (gymnosis) which makes these oligonucleotides attractive for therapeutic applications.We propose that LNAs provide a promising tool to develop a novel antiretroviral therapy targeting HIV-1 gene expression. Our preliminary results showed that gymnotically delivered LNAs, even more than transfected LNAs, display a very strong influence on the viral replication. Surprisingly, LNAs delivered unassisted, localize within the cytoplasm and here seem to induce degradation of viral mRNAs containing their target sequence. If the LNAs at a later time point also accumulate within the cell nucleus, and here disturbing viral alternative splicing by masking binding sites for splicing regulatory proteins, has to be analyzed. Therefore, we wish to analyze the cellular localization and distribution of gymnotically delivered LNAs in a time-course experiment and unravel the underlying LNA-mediated RNA degradation mechanisms (on-target effect). In addition, we want to investigate possible off-target effects including stimulation of innate immune responses. Furthermore, we aim at identifying additional LNA targets within the HIV-1 genome to develop an LNA cocktail as an alternative antiretroviral therapy.