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

交付的RNAi产品现已获得FDA批准，用于治疗由 影响肝脏中表达的基因的突变。基因载体携带的产生siRNAs的盒 对遗传性疾病有优势，因为有可能一次性治愈。我们解决了其中一个 AAV-U6 PolII中治疗性RNAi(转录RNAi)如何过表达的谜团 启动子驱动的shRNA(AAV-shRNA)引起急性肝毒性，并将继续对其进行更详细的研究。 当来自这样一个来源的siRNA达到或超过总miRNA读数的12%时，就有10% 第一个合成的miR122亚型(但不是其他miRNAs)减少，从而导致急性肝损伤 毒性表现为肝酶升高，在某些情况下会导致肝功能衰竭和死亡。因为生殖系 MiR122基因敲除的表型要小得多，我们假设这些结果中的不一致是 与miR122前体RNA转录本的差异表达有关，称为长非编码 RNA122(LNC122)，这两个RNA具有独立但协调的功能。我们建议 通过去除lnc122和miR122来阐明核定位的lnc122 rna的分子功能 然后将不同的单个RNA成分重新引入细胞、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lnc122染色质相互作用图。 这些研究很重要，因为miR122/lnc122基因不仅有肿瘤抑制因子 功能，但已知它也对正常肝再生、肝细胞形成有影响 癌症和肝纤维化与各种肝病，如NASH，脂代谢，和 病毒性肝炎感染。靶向肝脏外基因转移/表达的新策略包含 在3‘端非编码区以miR122为靶点的转基因，以排除在肝细胞中的泄漏表达。这就像是一些 在临床试验中测试的反义miR122产品，以及乙肝病毒和感染导致 MiR122的海绵生长及其长期影响尚不清楚。在授权期结束时，我们将 对lnc122-mir122产生的各种rna产物的功能有更深入的了解。 基因座及其在细胞内稳态中的作用，以及这可能如何有效地限制基于RNAi的治疗。 此外，随着我们更多地了解这个遗传位点的功能，它将为我们提供更多关于它是如何 参与所记录的疾病过程。这可能会为更优化的方法提供新的见解 治疗各种遗传病和获得性疾病的患者。

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.