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

描述（由申请人提供）：可以调节特定基因表达的小分子作为人类疾病的治疗试剂具有很大的潜力。吡咯-咪唑聚酰胺（PIPs）与DNA结合的特异性和亲和力与转录因子（TF）相当，具有细胞渗透性，定位于细胞核，甚至在染色质的背景下结合。在细胞培养系统中，PIPs已经被证明可以靶向启动子并影响大量基因的表达，并且一些研究已经证明了PIPs在半体内环境下治疗各种病理状况的功效。一项研究甚至在体内大鼠模型中显示了pip治疗慢性肾脏疾病的效用。然而，目前还没有关于pip用于治疗发育障碍或子宫内胚胎的研究报道。我们设计了靶向非编码RNA （ncRNA）的启动子区域kcnqot1的pip。这个长ncRNA的启动子是KvDMR1印迹控制区（ICR）的一部分，该区域调节小鼠远端7号染色体和人类11p15染色体上的一组印迹基因的表达。用这些PIPs处理小鼠胚胎成纤维细胞（mef）会导致kcnq10t1的转录沉默，并导致KvDMR1调节基因的正常沉默父本等位基因的表达。由于我们在该系统中的经验，我们选择了这种动物模型进行初步研究，以验证pip可以用于治疗子宫内发育障碍和其他疾病的假设。为了确定胚胎对PIP的摄取效率及其在子宫内的有效性，我们将首先在妊娠期间的不同时间点将荧光标记的PIP注射到携带野生型胚胎的妊娠小鼠的尾静脉中；然后将在显微镜下检查胚胎的PIP定位，并确定在体内施用PIP是否对7号染色体远端印迹表达有影响。接下来，我们建议挽救一个或所有kvdmr调节基因的母系遗传缺失的小鼠幼崽/胎儿的新生儿和妊娠中期死亡率。最后，我们将用pip治疗我们最近开发的人类过度生长条件的小鼠模型，Beckwith-Wiedemann综合征（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理证明，pip是体内和子宫内操纵基因表达的有效药物。pip可能最终提供一个新的基因特异性治疗试剂库，以对抗广泛的人类疾病。

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.