Acute/chronic limitations to transcriptional RNAi therapies for infectious and other liver diseases

递送的RNAi产品现已被FDA批准用于治疗由以下原因引起的两种遗传疾病： 影响肝脏中表达基因的突变。产生siRNA的基因载体递送盒 对遗传性疾病有优势，因为它有可能一次性治愈。我们解决了一个 如何从AAV-U6 polII过度表达治疗性RNAi（转录RNAi）的奥秘 启动子驱动的shRNA（AAV-shRNA）引起急性肝毒性，并继续对此进行更详细的研究。 当来自这种来源的siRNA达到总miRNA读段的12%或更多时， 第一个合成的miR 122同种型（而不是其他miRNAs）的减少，这诱导了急性肝损伤。 毒性的例子是肝酶升高，在某些情况下肝衰竭和死亡。因为生殖细胞 miR 122的敲除具有更少的表型，我们假设这些结果的不一致性是 与miR 122前体RNA转录物的差异表达相关，称为长非编码区。 RNA 122（lnc 122），这两种RNA具有独立但协调的功能。我们建议 阐明核定位的lnc 122 RNA的分子功能，并通过去除lnc 122和miR 122 然后将不同的单个RNA组分重新引入细胞、小鼠肝脏和 肝细胞癌模型这将使我们能够分离RNA的各个功能， 产品.我们还将绘制lnc 122染色质相互作用的图谱。 这些研究很重要，因为miR 122/lnc 122基因不仅具有肿瘤抑制因子， 功能，但它也被认为对正常的肝再生，肝细胞的形成有影响。 癌症和与各种肝脏疾病如NASH、脂质代谢和肝纤维化相关的肝纤维化。 病毒性肝炎感染靶向肝外基因转移/表达的新策略包括： 在3 'UTR中具有miR 122靶向的转基因以排除肝细胞中的渗漏表达。这就像一些 在临床试验中测试的反义miR 122产物，以及肝炎病毒B和感染导致 miR 122的海绵化及其长期影响尚不清楚。在资助期结束时，我们会 更好地理解由lnc 122-miR 122产生的各种RNA产物的功能 基因座及其在细胞内稳态中的作用，以及这如何有效地限制基于RNAi的治疗。 此外，随着我们对这一基因位点的功能了解得更多，它将为我们提供更多的见解， 参与所述的疾病过程。这可能为更优化的方法提供新的见解， 治疗各种遗传性和后天性疾病的患者。

Delivered RNAi products have now been FDA approved for treating two genetic disorders resulting from mutations affecting genes expressed in the liver. Gene vector delivered cassettes that produce siRNAs have an advantage for genetic disorders because of the potential for a one-shot cure. We solved one of the mysteries of how over expression of therapeutic RNAi (transcriptional RNAi) from an AAV-U6 polII promoter driven shRNA (AAV-shRNA) caused acute liver toxicity and continue to study this in more detail. When siRNAs from such a source reached 12% or more of the total miRNA reads there was a 10% reduction in the first synthesized miR122 isoform (but not the other miRNAs) and this induced acute liver toxicity exemplified by elevated liver enzymes and in some cases liver failure and death. Because germline knockout of miR122 has a much lesser phenotype, we hypothesize the discordance in these outcomes is related to the differential expression of the miR122 precursor RNA transcript known as long-non-coding RNA 122 (lnc122) and that these two RNAs have separate but coordinated functions. We propose to elucidate the molecular function of nuclear localized lnc122 RNA and by removing lnc122 and miR122 RNAs and then reintroducing the different individual RNA components in cells, mouse liver and hepatocellular carcinoma models. This will allow us to separate the individual functions of the RNA products. We will also map the lnc122 chromatin interactions. These studies are important because not only does the miR122/lnc122 gene have a tumor suppressor function, but it is also known to have effects on normal liver regeneration, formation of hepatocellular carcinoma, and liver fibrosis associated with various liver diseases such as NASH, lipid metabolism, and viral hepatitis infection. Newer strategies to target gene transfer/expression outside the liver contain transgenes with miR122 targets in the 3'UTR to exclude leaky expression in hepatocytes. This like some of the antisense miR122 products tested in clinical trials, and hepatitis virus B and infections result in the sponging of miR122 and the long-term effects of this are unclear. At the end of the granting period, we will have a better understanding of the function of the various RNA products produced from the lnc122-miR122 locus and their role in cellular homeostasis and how this may effectively limit RNAi based therapeutics. Moreover, as we learn more about the function of this genetic locus it will provide more insights into how it participates in the disease processes noted. This may provide new insights into more optimal means to treat patients with a variety of genetic and acquired diseases.