FAST is very excited to provide a summary of the newly published and incredibly promising manuscript titled, Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain[1] . This is work performed by Dr. David Segal and his laboratory at the Genome Center, MIND Institute, and Department of Biochemistry and Molecular Medicine at the University of California, Davis.  This work was funded in part by The Foundation for Angelman Syndrome Therapeutics (FAST).  Dr. Segal is a member of the FAST FIRE Team. 


This is an interpretation by Dr. Allyson Berent, Scientific Director of FAST, of the manuscript. This summary is not written by any of the authors of this scientific manuscript. Please review the prior summary on Angelman Syndrome in the FAST blog to re-introduce the genetic basics of Angelman Syndrome (AS). This will make it easier to follow each future discussion. 

Angelman Syndrome (AS) is a neurological genetic disorder caused by the loss of expression of the maternal copy of the gene UBE3A. Since the paternal copy, which is present and normal in individuals with AS, is imprinted in the neurons of the brain, it is essentially “turned off” or “silenced.” When the maternal copy is not present, as in the case of AS, there is an essential deficit of this gene’s functional end-products within the brain, and this results in the syndrome known as AS.

The paternal copy of this gene is “turned off” by something known as the “antisense transcript” or AT (UBE3A-AT), which is only present in the neurons of the central nervous system (CNS). Inhibiting the function of this antisense transcript may lead to unsilencing, or “turning on” of the paternal UBE3A gene, providing a therapeutic approach to restoring functional UBE3A in the CNS of those with Angelman Syndrome. This has been tried in laboratory animals with various different types of molecules and drugs (e.g. topoisomerase inhibitor drugs, antisense oligonucleotides [ASO], etc…), and partial unsilencing and some phenotypic rescue has been seen. The challenge with most therapeutics of this type trialed in animal models of AS has been attaining widespread delivery of the therapeutic throughout the brain so that a maximal effect can be achieved where it is needed, while concurrently avoiding undesirable side effects. In addition, this approach requires developing a delivery method that is safe and minimally invasive (e.g. spinal fluid injection, direct brain injection, etc…). In this report, a newly engineered artificial transcription factor (ATF), called a Zinc-finger based ATF, was used to silence the UBE3A-AT in a mouse model. The Zinc-finger based ATF, and other technology such as CRISPR (clustered regularly interspaced short palindromic repeat) and TALEs (transcription activator-like effectors), are all purified genetically engineered DNA binding proteins that can be designed as specifically as necessary. These factors can be programmed to affect gene expression by attaching to various areas on DNA. In AS, the UBE3A-AT on the paternal allele is the area of focus.

This study showed that Zinc-finger based ATF technology, when injected peripherally, meaning not directly into the brain or spinal fluid but rather into the subcutaneous tissue (under the skin) or peritoneal cavity (into the abdominal space surrounding the organs), was able to effectively cross the blood brain barrier (BBB). and provide Ube3a protein expression in a widespread distribution throughout the brain of genetically engineered mice with Angelman Syndrome. This means that the only way these mice would be able to get production of the Ube3a protein would be if the paternal UBE3A gene was activated to produce this protein. This activation supports that unsilencing the paternal UBE3A was successful using Zinc-finger based ATF technology. This technology not only “turned off the off switch,” but also provided widespread distribution throughout the brain with a simple peripheral injection!

This is the first report of using a peripherally injectable Zinc-finger protein that caused widespread activation of a gene within the brain, and the first report of this being done with any technology by peripheral administration, thus avoiding the need for direct brain or spinal fluid puncture. These findings will have very important implications in further explorations of treatment options for AS.

The blood brain barrier is made by a tight connection of cells within the blood vessels surrounding the CNS. This limits the method of delivery for most biologics, protecting the brain from exposure to systemic drugs or molecules. When it is desired for these therapeutics to get into the CNS, then, direct brain or spinal fluid injections may be needed. Previous approaches, like adeno-associated virus gene therapy, which required direct injection into various regions of the brain tissue, was able to show restoration of Ube3a in the brain of mice, but there was only a partial improvement seen in learning and memory, and no major change in motor coordination. This may be explained by the fact that there was limited distribution of the viral vectors throughout the brain tissue due to this direct injection. Widespread distribution, and crossing of the BBB through a peripheral injection, as seen with the Zinc finger based-ATF in this study, is an incredibly important accomplishment for future therapeutics.

An alternative delivery approach utilizing a penetrating peptide (HIV TAT), was explored in this study in order to accomplish delivery and distribution. This peptide is capable of delivering large proteins to the entire brain after a single peripheral injection. This was the case with both subcutaneous and intraperitoneal injections.

In this study, the ATF’s only provided transient gene regulation, suggesting they would need to be re-administered on a regular basis for long-term efficacy; therefore, peripheral subcutaneous injections would be a more reasonable therapeutic approach in the future.

Throughout the course of this 4-week study, no overt signs of toxicity were observed in the mice. There was a normal appearance and behaviors observed in the mice, no visual organ pathology on post-mortem examination was reported, and all regions of the brain showed widespread distribution of the ATFs.  Ube3a protein was found to be significantly increased in the AS mice treated with the TAT-Zn finger based-ATF (TAT-S1). The level of Ube3a protein increased to an intermediate level, between the AS-untreated and wild type control mice (mice unaffected by AS and not receiving treatment).  The cause for only an intermediate level of rescue may have been due to an insufficient concentration of the TAT-S1 protein at the target site. Future experiments with an increased dose or frequency of administration are needed.

In principle, the use of ATF biologics with short-term effects, similar to drugs with a short half-life, may have some advantages over more permanent therapeutic consideration where distribution, dosage and reversibility are currently problematic. One limitation of the current study was its short-term nature, as long-term immunity to these therapeutic proteins was not able to be studied; however, it is exciting that the development of the ATF approach could have a significant impact on Angelman Syndrome, and beyond. Other neurological disorders would only require small adjustments to the DNA-binding domain of the ATF used in this study in order to target other disease-related loci (e.g. Rett Syndrome or Prader-Willi, Huntington’s Disease, Pitt-Hopkins, and numerous autism spectrum disease genes).

Further research by Dr. Segal’s lab is currently underway to assess phenotypic rescue in the AS mouse model using this technology. This will more accurately give us an idea of the clinical impact this could have on individuals with AS. Other animal models will be considered if behavioral characteristics can be quantified allowing for changes to be observed.

[1] Bailus B, Pyles B, McAlister M, O’Green H, Lockwood S, Adams A, Tran Nguyen J, Yu A, Berman R, and Segal D., “Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain.” Mol Ther. 2016 Epub.