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

基因特异性小分子拯救子宫内发育障碍

基本信息

批准号：
8135228
负责人：
MICHAEL Joseph HIGGINS
金额：
$ 15.19万
依托单位：
ROSWELL PARK CANCER INSTITUTE CORP
依托单位国家：
美国
项目类别：
财政年份：
2010
资助国家：
美国
起止时间：
2010-09-01 至 2014-02-28
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8135228
关键词：
11p15 Affect Affinity Alleles Angiotensin II Animal Model Arteries Beckwith-Wiedemann Syndrome Binding Birth Boxing CDKN1C gene Cell Culture System Cell Culture Techniques Cell Nucleus Cell physiology Cells Chromatin Chromosomes Chromosomes, Human, Pair 7 Chronic Kidney Failure DNA Defect Development Diagnosis Disease Distal Dose Down-Regulation Embryo Endothelial Cells Engineering Fetus Fibroblasts Gene Expression Genes Growth Growth Suppressor Genes Human Imidazole Inherited Injury Label Lectin Low Density Lipoprotein Receptor Methylation Modeling Mus Mutation Neonatal Newborn Infant Nylons Patients Pharmaceutical Preparations Pilot Projects Pregnancy Pregnant Women Promoter Regions Pyrroles Rattus Reagent Reporting Repression Specificity Study Section System Tail Testing Therapeutic Time Tissues Toxic effect Untranslated RNA Veins design developmental disease experience fighting human disease imprint improved in utero in vivo mouse model mutant novel oxidized low density lipoprotein pregnant promoter public health relevance pup research study restenosis small molecule transcription factor uptake

项目摘要

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.

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（PIP）与DNA具有特异性和亲和力结合，可与转录因子（TF）相当，是细胞渗透性，位于细胞核中，甚至在染色质的背景下结合。在细胞培养系统中，PIP已被证明靶向启动子并影响大量基因的表达，几项研究表明，PIP在半成立体内环境中治疗各种病理条件方面的疗效。一项研究甚至表明，在体内大鼠模型中，PIP的效用用于治疗慢性肾脏疾病。但是，尚无研究报告使用PIP来治疗发育障碍或治疗子宫内胚胎的研究。我们设计了针对称为KCNQ1OT1的非编码RNA（NCRNA）的启动子区域的PIP。该长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染色体染色体中的印迹基因簇和11p15染色体的人类对应物的表达。用这些PIPs处理小鼠胚胎成纤维细胞（MEF）会导致KCNQ1OT1的转录沉默，并导致通常由KVDMR1调节的基因的正常沉默父亲等位基因的表达。由于我们在该系统中的经验，我们选择了该动物模型进行试点研究，以检验以下假设：PIP可用于治疗子宫内的发育障碍，也许还有其他疾病。为了确定PIP摄取到胚胎的效率，以及它们在子宫内的功效，我们将首先将荧光标记的PIP在妊娠期间在各个时间点携带野生型胚胎的怀孕小鼠的尾静脉标记为尾静脉；然后，将在显微镜下检查胚胎以进行PIP定位，并确定在体内施用的PIP是否对远端染色体染色体表达的影响有影响。接下来，我们建议用一个或所有KVDMR调节基因的母遗传缺失的小鼠幼崽/胎儿的新生儿和中期致死性。最后，我们将处理我们最近开发的人类过度生长条件的小鼠模型Beckwith-Wiedemann Syndome（BWS），并试图使这些小鼠的过度生长归一化。我们预计这些研究将提供原则上的证明，即PIP是在体内和子宫内操纵基因表达的有效药物。 PIPS最终可能会提供新型基因特异性治疗试剂的武器库，以与大量的人类疾病作斗争。公共卫生相关性：该应用建议测试一类新型的小分子，称为吡咯咪唑 - 咪唑多酰胺，因为它们能够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。这些研究将突出这些药物在治疗多种人类疾病方面的实用性。