Rescue of developmental disorders in utero by gene-specific small molecules

描述(申请人提供)：可以调节特定基因表达的小分子具有作为人类疾病治疗试剂的巨大潜力。吡咯咪唑多酰胺(PIP)与DNA的结合具有与转录因子(Tf)相当的特异性和亲和力，是细胞渗透性的，定位于细胞核，甚至结合在染色质的背景下。在细胞培养系统中，PIP被证明以启动子为靶点并影响大量基因的表达，一些研究已经证明PIP在半活体环境中治疗各种病理条件的有效性。一项研究甚至显示了PIPs在活体大鼠模型中用于治疗慢性肾脏疾病的效用。然而，还没有关于PIP用于治疗发育障碍或治疗子宫内胚胎的研究报道。我们设计了针对非编码RNA(NcRNA)的启动子区域的PIP，称为Kcnq1ot1。这一长ncRNA的启动子是KvDMR1印记控制区(ICR)的一部分，该区域调控小鼠7号染色体远端和人类11p15染色体上的一组印记基因的表达。用这些PIP处理小鼠胚胎成纤维细胞(MEF)会导致Kcnq1ot1的转录沉默，并导致KvDMR1调控基因的正常沉默的父系等位基因的表达。由于我们在这一系统中的经验，我们选择了这个动物模型进行试点研究，以检验PIP可以用于治疗发育障碍的假设，或许还有其他子宫内的疾病。为了确定PIP在胚胎中的摄取效率和在子宫内的有效性，我们首先将荧光标记的PIP注射到怀孕期间不同时间点携带野生型胚胎的怀孕小鼠的尾静脉；然后在显微镜下检查胚胎的PIP定位，并确定体内注射PIP是否对远端7号染色体印迹表达有影响。接下来，我们建议通过母系遗传缺失一个或所有KvDMR调节基因来挽救小鼠幼崽/胎儿的新生儿和妊娠中期死亡。最后，我们将用PIP治疗我们最近开发的人类过度生长状况的小鼠模型Beckwith-Wiedemann Syndome(BWS)，试图使这些小鼠的过度生长正常化。我们预计，这些研究将提供原则证明，即PIP是在体内和子宫内操纵基因表达的有效试剂。PIPS最终可能提供一批新型的基因特异性治疗试剂来对抗广泛的人类疾病。 公共卫生相关性：这项申请建议测试一类名为吡咯-咪唑多酰胺的新型小分子，以了解它们在小鼠胚胎发育中改变特定基因表达的能力，从而改善发育缺陷。这些研究将强调这些药物在治疗各种人类疾病方面的效用。

DESCRIPTION (provided by applicant): Small molecules that can modulate the expression of specific genes have great potential as therapeutic reagents for human disease. Pyrrole-Imidazole polyamides (PIPs) bind to DNA with specificities and affinities comparable to transcription factors (TF), are cell permeable, localize to the cell nucleus, and even bind in the context of chromatin. In cell culture systems, PIPs have been shown to target the promoters and affect the expression of a large number of genes, and several studies have demonstrated the efficacy of PIPs in treating various pathological conditions in semi-in vivo settings. One study even showed the utility of PIPs for the treatment of chronic kidney disease in an in vivo rat model. However, no studies have been reported where PIPs were used to treat developmental disorders, or to treat embryos in utero. We have designed PIPs that target the promoter region of the noncoding RNA (ncRNA) termed Kcnq1ot1. The promoter for this long ncRNA is part of the KvDMR1 imprinting control region (ICR) that regulates the expression of a cluster of imprinted genes in mouse distal chromosome 7 and the human counterpart at chromosome 11p15. Treatment of mouse embryonic fibroblasts (MEFs) with these PIPs causes the transcriptional silencing of Kcnq1ot1 and results in the expression of normally silent paternal alleles of genes regulated by KvDMR1. Because of our experience in this system, we chose this animal model for pilot studies to test the hypothesis that PIPs can be used to treat developmental disorders and perhaps other disease in utero. To determine both the efficiency of PIP uptake into embryos, and their efficacy in utero, we will first inject fluorescently labeled PIPs into the tail veins of pregnant mice carrying wild type embryos at various time points during gestation; embryos will then be examined microscopically for PIP localization and to establish whether PIP administered in vivo has effects on distal chromosome 7 imprinted expression. Next, we propose to rescue neonatal and mid-gestational lethality of mouse pups/fetuses with maternally inherited deletions of one or all KvDMR-regulated genes. Finally, we will treat our recently developed mouse model of the human overgrowth condition, Beckwith-Wiedemann syndome (BWS), with PIPs in an attempt to normalize excessive growth in these mice. We anticipate that these studies will provide proof-of-principle that PIPs are efficient agents for manipulating gene expression in vivo and in utero. PIPs may eventually provide an arsenal of novel gene-specific therapeutic reagents to fight a wide-range of human disease. PUBLIC HEALTH RELEVANCE: This application proposes to test a novel class of small molecules called pyrrole- imidazole polyamides for their ability to alter the expression of specific genes in developing mouse embryos thereby ameliorating developmental defects. These studies will highlight the utility of these drugs in the treatment of a wide variety of human diseases.