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

描述（由申请人提供）：可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巨大潜力。吡咯-咪唑聚酰胺（PIP）以与转录因子（TF）相当的特异性和亲和力与DNA结合，是细胞可渗透的，定位于细胞核，甚至结合在染色质的背景中。在细胞培养系统中，PIP已经显示出靶向启动子并影响大量基因的表达，并且几项研究已经证明PIP在半体内环境中治疗各种病理状况的功效。一项研究甚至显示PIP在体内大鼠模型中用于治疗慢性肾病的效用。然而，还没有研究报道PIP用于治疗发育障碍或治疗子宫内胚胎。我们设计了靶向称为Kcnq 1 ot 1的非编码RNA（ncRNA）启动子区的PIP。这种长ncRNA的启动子是KvDMR 1印记控制区（ICR）的一部分，该区域调节小鼠远端7号染色体和人类染色体11 p15处的印记基因簇的表达。用这些PIP处理小鼠胚胎成纤维细胞（MEF）导致Kcnq 1 ot 1的转录沉默，并导致由KvDMR 1调节的基因的通常沉默的父系等位基因的表达。由于我们在该系统中的经验，我们选择该动物模型进行初步研究，以测试PIP可用于治疗子宫内发育障碍和其他疾病的假设。为了确定PIP摄取到胚胎中的效率及其在子宫内的功效，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;然后将显微镜检查胚胎的PIP定位并确定体内施用的PIP是否对远端7号染色体印记表达具有影响。接下来，我们建议挽救新生儿和妊娠中期死亡的小鼠幼仔/胎儿与母系遗传缺失的一个或所有KvDMR调控基因。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。我们预期这些研究将提供PIP是用于操纵体内和子宫内基因表达的有效试剂的原理证明。PIP最终可能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。 公共卫生相关性：本申请提出测试一类称为吡咯-咪唑聚酰胺的新型小分子改变发育中的小鼠胚胎中特定基因的表达从而改善发育缺陷的能力。这些研究将突出这些药物在治疗各种人类疾病中的效用。

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.