Reactivation of MECP2 by Artificial Transcription Factors

Raf Schmid, PhD, MSCR | University of Pennsylvania

$141,912 AWARDED

Girls with Rett Syndrome have one mutated and one normal copy of the MECP2 gene in each brain cell, also known as neurons. These neurons randomly choose to utilize either the mutated or the normal copy of the MECP2 gene, and silence the other copy. If the normal MECP2 gene is silenced and the mutated MECP2 gene is active, then neurons become dysfunctional, which causes the typical disease symptoms. Studies in Rett mouse models have shown that restoring the normal MECP2 gene in neurons can reverse most Rett Syndrome symptoms, suggesting incredible therapeutic promise for activating of the normal MECP2 gene copy in human patients.

Researchers have been trying to activate the silenced copy of the MECP2 gene by using drugs, without much success. The investigator will now explore a novel approach using genetic tools that directly interact with the chromosome region that is responsible for switching the MECP2 gene on and off. Their proposed approach aims to flip the switch to turn on the silenced MECP2 gene copy without changing the genetic code itself. If successful in cell culture and mouse models, this strategy could lead to a transformative treatment for individuals suffering with Rett.

Gene expression is regulated by genetic elements directly adjacent to the gene itself. This chromosomal region is called the promoter, which functions as an off/on switch for the adjacent gene. In diseased Rett neurons the promoter of the normal MECP2 gene copy is kept in the “off”, or silenced, state by certain modifications that prevent binding of proteins, called transcription factors, that could facilitate activation of the MECP2 gene. The lab proposes a novel approach to activate the silenced MECP2 gene by utilizing special DNA-binding proteins, called zinc finger proteins, which selectively bind to the MECP2 promoter region. Zinc finger proteins were originally developed over a decade ago for the first attempts of gene editing. Investigators will repurpose zinc finger proteins and utilize them to target an artificial transcription factor to the MECP2 promoter to override the silenced state and jump-start gene activation.

The goal is to selectively switch on the MECP2 gene by this approach and restore normal MeCP2 protein to all neurons. The lab will design, manufacture and test a number of possible zinc finger proteins and different artificial transcription factors to find the required optimal combination for MECP2 activation. In order to test a maximum amount of combinations, experiments will initially be carried out in cultured mouse neurons. Leads will subsequently be tested in Rett mouse models. Ultimately, promising zinc finger/artificial transcription factor complexes will need to be delivered to the brain. Vectors being developed by our Gene Therapy Consortium can be easily adapted to enable delivery.

RSRT is also funding the lab of Rudolf Jaenisch to use CRISPR technology to reactivate MECP2. It’s critical that we pursue multiple approaches to reactivating MECP2 in parallel. There are certain advantages to each approach. One advantage of zinc finger proteins is that they are much smaller compared to the currently used CRISPR complexes. The chromosome regions that include the silenced MECP2 promoter and gene are highly compacted so the smaller zinc finger protein complex may have an advantage over CRISPR. Additionally, to date, CRISPR technology has only been used in clinical trials and FDA-approved applications outside of the human body (called ex-vivo), to modify cells in a dish for re-injection. However, since the target organ for Rett is the brain, an ex-vivo approach would not work. In the case of Rett the gene activation tools have to be delivered into the brain. The only brain-penetrant delivery technology that is currently being tested in clinical trials is adeno-associated virus (AAV). Zinc finger/artificial transcription factor complexes can be delivered more easily via AAV than the larger CRISPR.

We are excited about the promise of both the zinc finger and CRISPR strategies for reactivating MECP2 and look forward to keeping you updated.


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