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Dose-Dependent Nuclear Delivery and Transcriptional Repression with a Cell-Penetrant MeCP2.

Xizi ZhangClaudia CattoglioMadeline ZoltekCarlo VetrallaDeepto MozumdarAlanna Schepartz
Published in: ACS central science (2023)
The vast majority of biologic-based therapeutics operate within serum, on the cell surface, or within endocytic vesicles, in large part because proteins and nucleic acids fail to efficiently cross cell or endosomal membranes. The impact of biologic-based therapeutics would expand exponentially if proteins and nucleic acids could reliably evade endosomal degradation, escape endosomal vesicles, and remain functional. Using the cell-permeant mini-protein ZF5.3, here we report the efficient nuclear delivery of functional Methyl-CpG-binding-protein 2 (MeCP2), a transcriptional regulator whose mutation causes Rett syndrome (RTT). We report that ZF- t MeCP2, a conjugate of ZF5.3 and MeCP2(Δaa13-71, 313-484), binds DNA in a methylation-dependent manner in vitro , and reaches the nucleus of model cell lines intact to achieve an average concentration of 700 nM. When delivered to live cells, ZF- t MeCP2 engages the NCoR/SMRT corepressor complex, selectively represses transcription from methylated promoters, and colocalizes with heterochromatin in mouse primary cortical neurons. We also report that efficient nuclear delivery of ZF- t MeCP2 relies on an endosomal escape portal provided by HOPS-dependent endosomal fusion. The Tat conjugate of MeCP2 (Tat- t MeCP2), evaluated for comparison, is degraded within the nucleus, is not selective for methylated promoters, and trafficks in a HOPS-independent manner. These results support the feasibility of a HOPS-dependent portal for delivering functional macromolecules to the cell interior using the cell-penetrant mini-protein ZF5.3. Such a strategy could broaden the impact of multiple families of biologic-based therapeutics.
Keyphrases
  • single cell
  • cell therapy
  • rheumatoid arthritis
  • binding protein
  • transcription factor
  • dna methylation
  • stem cells
  • cell death
  • spinal cord
  • bone marrow
  • cell surface
  • oxidative stress
  • cell cycle arrest
  • genome wide