Protein Delivery of an Artificial Transcription Factor Restores Widespread Ube3a Expression in an Angelman Syndrome Mouse Brain

Quick Overview

Researchers have developed an artificial transcription factor that can restore widespread Ube3a expression in the brain of a mouse with Angelman Syndrome. The team used a cell-penetrating peptide from HIV to deliver the protein into the brain. The protein was tracked using a red fluorescent protein, mCherry, which allowed the team to observe its distribution throughout the brain. The protein was found to activate the epigenetically silenced paternal allele in the mouse brain, leading to a significant increase in Ube3a. The team is now working on testing off-target effects and immune responses, as well as evaluating alternative delivery methods.

Introduction

Have you ever wondered how scientists determine the effectiveness of a protein in a study? In this case, we examined four other proteins and their combinations. Interestingly, we found that the S100 protein we initially chose was highly effective in repressing transcription at the promoter.

Protein Selection and Analysis

We analysed the SNURF–SNRPN expression in Neuro2a cells using RT-qPCR. The S100 protein significantly repressed transcription at the promoter, while the other four zinc fingers we created did not. In fact, some seemed to activate transcription, and their combinations activated even more.

Protein Delivery into the Brain

To apply this as a therapy, we needed a way to deliver this protein into the brain. We considered several approaches, including viral vectors, but ultimately chose to use a cell-penetrating peptide from HIV. This peptide supposedly allows its cargo to cross membranes, including the blood-brain barrier.

Tracking the Protein

We attached a red fluorescent protein, mCherry, to the protein, which allowed us to track it easily. We could even track it in live animals using in vivo fluorescence imaging devices. We found that the protein distributed widely throughout the brain, which was a positive outcome we hadn’t predicted.

Testing UBE3A Activation

We then tested whether the protein could activate UBE3A. We treated animals for four weeks with three injections per week and found a significant increase in UBE3A. We also saw some isoforms of UBE3A on a Western blot.

Remaining Challenges and Future Steps

While we’ve shown a molecular rescue of UBE3A expression, we need to understand its effects on the mice and whether it rescues phenotypes. We’re also testing for off-target effects and immune responses. We’re considering alternative delivery methods and ways to increase persistence and delivery.

Conclusion

Our study showed that we could design an artificial transcription factor that could cross the blood-brain barrier, enter neurons throughout the brain, and activate the epigenetically silenced paternal allele in the mouse brain. We’re grateful for the support and inspiration from the Angelman Syndrome Foundation and the Foundation for Angelman Syndrome Therapeutics.

Questions and Answers

During the presentation, several questions were asked about the choice of the repression transcription factor to activate UBE3A, the activation seen with other proteins, and the use of CRISPR-Cas. The author clarified that the S100 protein was chosen for its effectiveness, the activation seen with other proteins is not fully understood, and CRISPR-Cas was not used due to the injectable approach of the study.