While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from th...While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference(RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by noncoding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing(TGS), as well as finetuning their expression through post-transcriptional gene silencing(PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.展开更多
Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing comple...Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA- targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA- specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells’ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).展开更多
In the last decade, RNA interference(RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased ...In the last decade, RNA interference(RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased inhibitors against the chronic infection with human immunodeficiency virus type 1(HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules(sh RNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 sh RNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three sh RNAs. These sh RNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome,exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial sh RNA vector will soon move forward to the first clinical studies.展开更多
目的利用小干扰RNA(small interfering RNA,si RNA)片段沉默卵巢癌细胞人垂体瘤转化基因1(human pituitary tumor-transforming gene 1,h PTTG1)基因表达,探讨其抑制细胞凋亡的分子机制。方法通过脂质体将h PTTG1 si RNA转染A2780细胞(h...目的利用小干扰RNA(small interfering RNA,si RNA)片段沉默卵巢癌细胞人垂体瘤转化基因1(human pituitary tumor-transforming gene 1,h PTTG1)基因表达,探讨其抑制细胞凋亡的分子机制。方法通过脂质体将h PTTG1 si RNA转染A2780细胞(h PTTG1 si RNA干扰组),并设立正常组和阴性对照组,转染48 h后进行检测。采用实时荧光定量聚合酶链反应检测转染前后h PTTG1 m RNA表达水平的变化,采用逆转录-聚合酶链反应和蛋白质印迹法检测survivin m RNA和蛋白表达水平,采用DNA梯带电泳法和碘化丙啶单染法分析细胞凋亡,采用比色法测定胱天蛋白酶(caspase)-3活性。结果 h PTTG1 si RNA可抑制A2780细胞内h PTTG1 m RNA表达;h PTTG1 si RNA干扰组可见典型的细胞凋亡阶梯状电泳,流式细胞仪检测该组细胞凋亡率为(17.53±2.17)%,明显高于正常组和阴性对照组[(8.97±1.56)%、(9.64±1.31)%],差异有统计学意义(P<0.05);h PTTG1干扰后survivin m RNA和蛋白表达均下调,caspase-3活性增强。结论 h PTTG1 si RNA可下调survivin基因表达,活化caspase-3,导致A2780细胞凋亡,其可成为卵巢癌基因治疗的潜在靶点。展开更多
文摘While human immunodeficiency virus 1(HIV-1) infectionis controlled through continuous, life-long use of a combination of drugs targeting different steps of the virus cycle, HIV-1 is never completely eradicated from the body. Despite decades of research there is still no effective vaccine to prevent HIV-1 infection. Therefore, the possibility of an RNA interference(RNAi)-based cure has become an increasingly explored approach. Endogenous gene expression is controlled at both, transcriptional and post-transcriptional levels by noncoding RNAs, which act through diverse molecular mechanisms including RNAi. RNAi has the potential to control the turning on/off of specific genes through transcriptional gene silencing(TGS), as well as finetuning their expression through post-transcriptional gene silencing(PTGS). In this review we will describe in detail the canonical RNAi pathways for PTGS and TGS, the relationship of TGS with other silencing mechanisms and will discuss a variety of approaches developed to suppress HIV-1 via manipulation of RNAi. We will briefly compare RNAi strategies against other approaches developed to target the virus, highlighting their potential to overcome the major obstacle to finding a cure, which is the specific targeting of the HIV-1 reservoir within latently infected cells.
文摘Small interfering RNA (siRNA) and microRNA (miRNA) are small RNAs of 18-25 nucleotides (nt) in length that play important roles in regulating gene expression. They are incorporated into an RNA-induced silencing complex (RISC) and serve as guides for silencing their corresponding target mRNAs based on complementary base-pairing. The promise of gene silencing has led many researchers to consider siRNA as an anti-viral tool. However, in long-term settings, many viruses appear to escape from this therapeutical strategy. An example of this may be seen in the case of human immunodeficiency virus type-1 (HIV-1) which is able to evade RNA silencing by either mutating the siRNA- targeted sequence or by encoding for a partial suppressor of RNAi (RNA interference). On the other hand, because miRNA targeting does not require absolute complementarity of base-pairing, mutational escape by viruses from miRNA- specified silencing may be more difficult to achieve. In this review, we discuss stratagems used by various viruses to avoid the cells’ antiviral si/mi-RNA defenses and notions of how viruses might control and regulate host cell genes by encoding viral miRNAs (vmiRNAs).
基金Supported by The NWO-CW(Chemical Sciences),Zon Mw(Medical Sciences),the Dutch AIDS Fund(project 2006006)the DAAD(German Academic Exchange Service)the FRM(Fondation pour la Recherche Medicale)
文摘In the last decade, RNA interference(RNAi) advanced to one of the most widely applied techniques in the biomedical research field and several RNAi therapeutic clinical trials have been launched. We focus on RNAibased inhibitors against the chronic infection with human immunodeficiency virus type 1(HIV-1). A lentiviral gene therapy is proposed for HIV-infected patients that will protect and reconstitute the vital immune cell pool. The RNAi-based inhibitors that have been developed are short hairpin RNA molecules(sh RNAs), of which multiple are needed to prevent viral escape. In ten distinct steps, we describe the selection process that started with 135 sh RNA candidates, from the initial design criteria, via testing of the in vitro and in vivo antiviral activity and cytotoxicity to the final design of a combinatorial therapy with three sh RNAs. These sh RNAs satisfied all 10 selection criteria such as targeting conserved regions of the HIV-1 RNA genome,exhibiting robust inhibition of HIV-1 replication and having no impact on cell physiology. This combinatorial sh RNA vector will soon move forward to the first clinical studies.
文摘目的利用小干扰RNA(small interfering RNA,si RNA)片段沉默卵巢癌细胞人垂体瘤转化基因1(human pituitary tumor-transforming gene 1,h PTTG1)基因表达,探讨其抑制细胞凋亡的分子机制。方法通过脂质体将h PTTG1 si RNA转染A2780细胞(h PTTG1 si RNA干扰组),并设立正常组和阴性对照组,转染48 h后进行检测。采用实时荧光定量聚合酶链反应检测转染前后h PTTG1 m RNA表达水平的变化,采用逆转录-聚合酶链反应和蛋白质印迹法检测survivin m RNA和蛋白表达水平,采用DNA梯带电泳法和碘化丙啶单染法分析细胞凋亡,采用比色法测定胱天蛋白酶(caspase)-3活性。结果 h PTTG1 si RNA可抑制A2780细胞内h PTTG1 m RNA表达;h PTTG1 si RNA干扰组可见典型的细胞凋亡阶梯状电泳,流式细胞仪检测该组细胞凋亡率为(17.53±2.17)%,明显高于正常组和阴性对照组[(8.97±1.56)%、(9.64±1.31)%],差异有统计学意义(P<0.05);h PTTG1干扰后survivin m RNA和蛋白表达均下调,caspase-3活性增强。结论 h PTTG1 si RNA可下调survivin基因表达,活化caspase-3,导致A2780细胞凋亡,其可成为卵巢癌基因治疗的潜在靶点。
