MECP2 Reactivation
This promising strategy aims to cure Rett by activating a backup copy of MECP2 present in all female cells.
Our initial efforts at MECP2 reactivation were focused on finding small-molecule drugs that could achieve the desired effect. Scientists designed cell lines that could detect MECP2 gene reactivation, then tested hundreds of thousands of compounds using high-throughput robotic equipment. RSRT-funded scientists involved in this effort include Dr. Benjamin Philpot, PhD, Dr. Bryan Roth, MD, PhD, and Dr. Terry Magnuson, PhD, at the University of North Carolina at Chapel Hill; Dr. Jeannie Lee, PhD, at Harvard; Dr. Antonio Bedalov, MD, PhD, at the Fred Hutchinson Cancer Research Institute; Dr. Michael Green, MD, PhD, at the University of Massachusetts; Professor Joost Gribnau at Erasmus in the Netherlands; and Dr. Andrea Cerase, PhD, from the Queen Mary University of London.
Biologic Solutions to Reactivation
In recent years researchers from around the world generated additional data that made it possible to better understand how the X chromosome becomes inactivated. This additional data made it possible to begin designing biologic strategies for MECP2 reactivation. These strategies necessitate the ability to, first, target the specific spot on the X chromosome where the inactive MECP2 resides and, second, be able to bring molecules that can remove certain chemical tags from MECP2 that keep it silenced. The hope is to be able to effectively and efficiently deliver this therapeutic to the brain in a "one-and-done" manner.
RSRT has invested more than $7 million in MECP2 reactivation. RSRT-driven collaborations, initiatives, and investments have helped to generate all of the following MECP2 reactivation programs.
Alcyone Therapeutics
In March 2021 Alcyone Therapeutics announced their lead program, ACTX-101, a genetic strategy to reactivate the silenced MECP2 on the inactive X chromosome. Alcyone has partnered with two scientists, Dr. Sanchita Bhatnagar, PhD, and Dr. Kathrin Meyer, PhD, who began their Rett efforts with RSRT funding. Dr. Bhatnagar first started working on Rett as a post-doc in the RSRT-funded lab of Dr. Michael Green at University of Massachusetts. She continued that work when she began her independent career at University of Virginia. Dr. Bhatnagar has discovered that inhibiting certain microRNAs can lead to activation of the silenced MECP2 gene.
Dr. Meyer was a senior post-doc in the lab of Dr. Brian Kaspar who was part of the original RSRT Gene Therapy Consortium. When Dr. Kaspar left his academic post, Dr. Meyer took over his lab and continued working on Rett with RSRT funding.
RSRT introduced Drs. Bhatnagar and Meyer in 2018, and they developed a dynamic collaboration that has led to Alcyone licensing their discoveries. Furthermore, Alcyone has a novel delivery approach that should increase distribution of the therapeutic across the brain.
Kyle Fink and Antonio Bedalov
Inactivation of the MECP2 gene on the silent X chromosome is maintained by several biological mechanisms. Two key mechanisms are the presence of an RNA called XIST, which surrounds the inactive X chromosome like a cloud, and DNA methylation marks on the inactive X chromosome. The Fink and Bedalov labs have obtained evidence that removal of DNA methylation marks, as well as the XIST RNA from the inactive X chromosome, has the potential to robustly turn on the silenced copy of MECP2.
The labs are employing CRISPR-Cas9 fused to an enzyme called TET1 that removes DNA methylation marks from the MECP2 gene. Prior work in the Fink laboratory has successfully used this epigenome editing approach to activate a silenced copy of CDLK5 on the inactive X chromosome in vitro as a potential treatment strategy for CDKL5-deficiency disorder, a genetic disorder that is similar to Rett syndrome. In addition, the scientists will combine this targeted approach with global down-regulation of XIST. The Bedalov lab has already shown that XIST deletion in the mouse brain is well tolerated and reactivates MECP2. Building on the expertise of the Fink and Bedalov laboratories, parallel studies are being pursued in human and mouse cells in vitro and in mouse Rett syndrome models in vivo by delivery of epigenetic editors via adeno-associated viral vectors, paving the way for future clinical trials in girls with Rett syndrome.
Kyle Fink's lab is at the University of California, Davis and Antonio Bedalov's lab is at the Fred Hutchinson Cancer Research Institute.
Shawn Liu
It is a well known fact that about 20% of the genes on the inactive human X chromosome escape inactivation and are actively making protein. A common feature of these "escapee" genes is the unique physical structure of the DNA that surrounds them. The hypothesis is that the physical structure of the surrounding DNA helps to maintain gene activation.
Shawn Liu, PhD, theorized that by mimicking the DNA structure around the MECP2 gene he might coax the gene to stay active. He created the desired DNA structure by recruiting a protein called CTCF using CRISPR technology. He proposes a two-prong approach to MECP2 reactivation by combining the removal of methyl groups with the changing of the DNA structure around MECP2.
RSRT awarded funding to the Liu lab in 2022 to join the Bedalov/Fink collaboration. Each scientist brings unique resources to the collaboration – Liu brings the novel two-prong approach, Bedalov brings the mouse model he developed specifically for MECP2 reactivation experiments, and Fink brings the AAV packaging expertise.
Shawn Liu began his work on Rett as a post-doc in the RSRT-funded lab of Rudolf Jaenisch. In 2020 he transitioned to his own independent lab at Columbia University.
Rudolf Jaenisch
Several years ago we began funding the lab of Dr. Rudolf Jaenisch, PhD, to pursue a novel approach to reactivation that leverages CRISPR technology to deliver certain epigenetic molecules to the inactive MECP2. The epigenome controls when genes are turned on or off by adding or removing chemical tags. One can think of the genome (DNA) as a charm bracelet and the epigenome as charms that can be added and removed. Methyl tags on DNA keep genes silent and acetyl tags keep genes active. The video below explains the experiments being pursued in the Jaenisch lab.
It’s important to note that biologic therapeutics will need to be delivered to the brain, and therefore our ongoing delivery efforts will be highly relevant to our reactivation program.
Herophilus
In January 2021, Herophilus, Inc. announced its SB-9 program, a small-molecule therapy designed to reactivate the silent MECP2 gene on the inactive X chromosome. The SB-9 program was discovered using Herophilus’s AI-driven brain organoid drug discovery platform (the “OrCA Platform”) and Rett patient-derived stem cells provided by RSRT. By combining the biological richness of human brain organoid models, industrial automation, and advanced analytical techniques, Herophilus has demonstrated that the SB-9 molecule is able to both reactivate MECP2 at safe levels and reverse disease biology in human in vitro models of Rett syndrome.
Dr. Andrea Cerase, PhD
In this project Dr. Cerase of Queen Mary University of London aims to find small-molecule drugs that can reactivate MECP2 by deactivating the master regulator Xist. The scientists have generated reporter cell lines that can be used for unbiased, high-throughput drug screening using a 20,000 chemical library of peptide-mimetic that can cross the blood brain barrier. Drugs identified during the screen will then be tested in mouse models of Rett syndrome.