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

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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.