Invented by Benjamin David Philpot, Mark John Zylka, Bryan Leo Roth, John Arthur Allen, Hsien-Sung Huang, University of North Carolina at Chapel Hill

The market for methods and compositions to unsilencing imprinted genes is gaining significant attention in the field of genetic research and therapeutics. Imprinted genes are a unique subset of genes that are expressed in a parent-of-origin-specific manner, meaning that they are only active when inherited from one parent and silenced when inherited from the other parent. This phenomenon plays a crucial role in normal development and growth, but when imprinted genes are dysregulated, it can lead to various genetic disorders and diseases. The concept of unsilencing imprinted genes has emerged as a promising avenue for potential therapeutic interventions. By reactivating the silenced genes or silencing the active ones, researchers aim to restore the normal gene expression patterns and alleviate the associated health complications. This approach holds great potential for treating disorders such as Angelman syndrome, Prader-Willi syndrome, Beckwith-Wiedemann syndrome, and many others that are caused by imprinted gene dysregulation. Several methods and compositions have been developed to target imprinted genes and modify their expression. One of the most widely used techniques is the use of small interfering RNAs (siRNAs) or antisense oligonucleotides (ASOs) to specifically target and degrade the RNA molecules responsible for silencing the imprinted genes. These molecules can be designed to be highly specific, ensuring minimal off-target effects. Another approach involves the use of epigenetic modifiers, such as DNA methyltransferase inhibitors or histone deacetylase inhibitors, to alter the epigenetic marks on imprinted genes. These modifications can lead to the reactivation of silenced genes or the silencing of active ones. This method has shown promising results in preclinical studies and holds potential for future therapeutic applications. Furthermore, genome editing technologies, such as CRISPR-Cas9, are being explored to directly modify the DNA sequences of imprinted genes. By introducing specific mutations or deletions, researchers can potentially correct the underlying genetic defects responsible for imprinted gene dysregulation. The market for methods and compositions to unsilencing imprinted genes is expected to grow significantly in the coming years. The increasing understanding of the molecular mechanisms underlying imprinted gene regulation, coupled with advancements in gene editing technologies and drug delivery systems, is driving the development of novel therapeutic approaches. Pharmaceutical companies and biotechnology firms are actively investing in research and development in this field. Collaborations between academia and industry are fostering innovation and accelerating the translation of scientific discoveries into clinical applications. Additionally, the growing interest from venture capitalists and private investors indicates the market’s potential for commercial success. However, several challenges need to be addressed to fully exploit the therapeutic potential of unsilencing imprinted genes. The delivery of therapeutic molecules to target tissues and cells remains a significant hurdle. Developing efficient and safe delivery systems that can specifically target imprinted genes in a tissue-specific manner is crucial for successful clinical translation. Moreover, the long-term safety and efficacy of these interventions need to be thoroughly evaluated through rigorous preclinical and clinical studies. Understanding the potential off-target effects and unintended consequences of unsilencing imprinted genes is essential to ensure patient safety and avoid any unforeseen complications. In conclusion, the market for methods and compositions to unsilencing imprinted genes is a rapidly growing field with immense potential for therapeutic interventions. The development of novel techniques, such as siRNAs, epigenetic modifiers, and genome editing technologies, is paving the way for targeted and precise treatments for various genetic disorders caused by imprinted gene dysregulation. With continued research and investment, these advancements hold promise for improving the lives of individuals affected by imprinted gene-related diseases.

The University of North Carolina at Chapel Hill invention works as follows

The present invention provides compositions and methods for causing expression of Ube3a by contacting a cell with a Topoisomerase Inhibitor. One embodiment is a treatment for Angelman syndrome or other genomic imprinting disorders by administering a topoisomerase inhibitor to a patient.

Background for Methods and compositions to unsilencing imprinted gene

Angelman syndrome is a form of autism spectrum disorder that has no treatment. Angelman syndrome is characterized by a seemingly normal first year of development, followed by severe intellectual disabilities. These include seizures, EEG abnormalities and gait disorders, as well as profound language impairments. The deficits can be modelled in Ube3a deficient mice. These deficits result from maternal mutations or deletions of the E3-ubiquitin ligase Ube3a gene. The paternal allele is silenced by epigenetic imprinting in the majority of neurons, so the loss of function of the mother’s allele results in the elimination of Ube3a expression in neurons. 1). The health care costs associated with Angelman syndrome, even if you ignore the human costs, are enormous.

Genetic engineering was used to correct the neurological deficits of Angelman syndrome mice model (van Woerden, et. al., 2007, but it relies on knocking genes out, which makes it unpractical for humans. Pharmacological treatment is a viable alternative. Only one human clinical trial has tested the effects on methyl donors, betaine and folate (Arn, et. al. 1998; Bacino, et. al. 2003), but it hasn’t been successful.

The present invention provides compositions and methods for unsilencing genes that have been imprinted (e.g. the paternal gene of Ube3a, which has been silenced by epigenetic imprinting), thus providing treatment methods for genomic imprinting disorders such as Angelman Syndrome.

In one aspect, this invention provides a way to induce expression of Ube3a within a cell by contacting it with a sufficient amount of topoisomerase inhibitor, and thereby causing expression of Ube3a.

Another aspect of the invention is a treatment method for a genomic imprinting disease in a patient, which involves administering an effective amount to the patient of a topoisomerase inhibitor, thus treating the genomic imprinting condition in the patient.

Further aspects” of the invention include a treatment method for a disorder that is associated with epigenetic modifications in a patient, which involves administering an effective dose of topoisomerase inhibitor to the patient, thus treating the condition associated with epigenetic modifications in the patient.


FIG. 1. “Schema of treatment for Angelman syndrome by restoring the paternal Ube3a gene.

FIG. 2. Schematic of the drug discovery process to identify Angelman Syndrome therapeutics. A fluorescence assay is used to assess the unsilencing of paternal Ube3a. Fluid handling robots are used to treat 7 DIV cultures in quadruplicate using 10?M or 0.2% vehicle DMSO. The YFP signal was amplified using an anti-GFP (which also recognizes YFP), and the Ube3a – YFP fluorescence was assessed in >1200 neurons/well, 72 hr after drug or DMSO treatment.

FIG. 4. Irinotecan, a topoisomerase inhibitor, is able to unsilence paternal Ube3a. Panel A: Paternal Ube3a YFP is not expressed in neurons cultured with 0.2% DMSO. Panel B: Treatment of cultures with 10 M irinotecan for 72 hr turns on paternal YFP. This was independently replicated in n=10 separate experiments. Each experiment was run in quadruplicate on different days. The density of neurons and their health are similar between cells treated with vehicle and those treated with drug, as shown by counterstaining the nuclei marker DAPI.

FIG. 5. Additional assays confirm irinotecan’s ability to unsilence the paternal Ube3a – YFP allele. Irinotecan (10 M for 72 hrs.) increased the paternal Ube3a – YFP allele, as assessed by quantitative RTPCR experiments (Panel. A) and Western blots (Panel. B). Samples of separate cultures treated with vehicle; samples from separate cultures treated with drug. *p0.05 unpaired t test.

FIG. 6. Irinotecan is able to unsilence the paternal Ube3a gene in Angelman syndrome mouse model neurons. Irinotecan (10?M) increased Ube3a levels in neurons cultured for 72 hours from Ube3am+/p+ mouse. Levels reached?50% (in Ube3am+/p+). V=vehicle-treated; D=drug-treated. *p0.01, unpaired T-test.

FIG. 8. Irinotecan or topotecan may be able to unsilence paternal Ube3a. In (Panel a), the paternal Ube3a YFP allele is not expressed in spinal neurons. Injections of (Panel A) irinotecan (or (Panel D) topotecan intrathecally into living mice unmuted the paternal Ube3a YFP allele. These results were reproduced by n>4 mouse. The pan-neuronal NeuN marker was used to counterstain the neurons of mice treated with vehicle and drugs. Scale bar=100 ?m.

FIG. 9. Topotecan is effective in restoring paternal Ube3a to the entire brain after a single week of treatment. (Panel A1) Coronal section showing that intracerebroventricular osmotic minipump infusions of vehicle do not unsilence paternal Ube3a-YFP. The boxes highlight the regions of (Panel) A2 neocortex (and (Panel) A3) hippocampus at higher magnifications. Topotecan (Panel A1) for a week un-silences paternal Ube3a in the brain. In (Panel) B2, the neocortex, and (Panel) B3, the hippocampus are shown at higher magnifications. Panel C: Control labeling for maternal Ube3aYFP. This coronal section does not show some of the structures that are shown in Panel A1 and Panel B1, such as the hippocampus.

FIG. 10. The effects of topotecan on paternal Ube3a are long-lasting. Two weeks of daily intravenous therapy (Panel a) Topotecan injections (100 nmol/day for 10 out of 14 days) unmute paternal Ube3aYFP in some neurons (compare with more complete paternal unmuting at higher drug concentrations shown in FIG. 9, Panel C). Panel B: Unsilencing of the paternal Ube3a continues 14 days after the cessation topotecan infusions. This suggests that imprinting is not permanent. Panels C-D: Counterstaining Ube3a YFP with NeuN, the pan-neuronal marker. Scale bar=100 ?m. These experiments were carried out in mice that expressed Ube3a – YFP on both the maternal and paternal alleles.

FIG. 11. Ube3a deficient mice show deficits in reverse learning of the water-maze. In reversal-learning, significant genotype effects were observed [main effect, F(1)=7.47; genotype X location interaction,F(3)=6.29; p=0.0009]. In both groups, repeated measures within-genotype ANOVAs revealed target selection during acquisition [wildtype F(3,27), p0.0001 and m?/p+ F(3,30), p0.0001]. Only the wildtype group showed a significant preference to the new platform [F(3,27), p0.0001]. *p