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

在HIV-1复制过程中，病毒(+)RNA基因组被逆转录，转化为双链DNA，并整合到宿主细胞基因组中。前病毒的转录产生了一个多顺反子前mRNA，它以剪接或非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。由于病毒严重依赖于这些剪接和非剪接的mRNAs的平衡表达，这一比例的任何干扰都会极大地影响HIV 1的感染性和致病机制。最近，我们可以证明HIV-1基因组中的两个保守的剪接调控元件，GI3-2和ESEtat元件在病毒mRNAs的产生中发挥重要作用，如vif，vpr和tat mRNAs。此外，这些元件的突变抑制了病毒的复制。关于针对HIV-1基因表达的替代抗逆转录病毒疗法的开发，我们用反义寡核苷酸掩盖了这些剪接调控元件，也导致了HIV-1颗粒的产生。作为寡核苷酸，我们使用锁定核酸(LNAs)，这是由于额外的亚甲基桥而被限制在Watson-Crick结合的理想构象中的修饰核苷酸。除了与其他反义寡核苷酸相比的许多优点外，LNAs还可以在没有转染剂的情况下进入细胞内，这使得这些寡核苷酸具有治疗应用的吸引力。我们认为LNAs为开发针对HIV-1基因表达的新型抗逆转录病毒治疗提供了一种有前途的工具。我们的初步结果表明，体液输送的LNAs，甚至比转基因的LNAs，对病毒复制显示出非常强烈的影响。令人惊讶的是，LNAs是在没有辅助的情况下传递的，定位于细胞质中，在这里似乎可以诱导包含其目标序列的病毒mRNAs的降解。如果在稍后的时间点LNAs也在细胞核内积累，并在这里通过掩盖剪接调节蛋白的结合位点来干扰病毒的选择性剪接，则必须进行分析。因此，我们希望在时间过程实验中分析裸露递送的LNA的细胞定位和分布，并揭示LNA介导的RNA降解机制(靶上效应)。此外，我们还想研究可能的非靶点效应，包括刺激先天免疫反应。此外，我们的目标是在HIV-1基因组中确定更多的LNA靶点，以开发LNA鸡尾酒作为一种替代的抗逆转录病毒疗法。

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.