Targeted Reactivation of FMR1 Transcription in Fragile X Syndrome Embryonic Stem Cells

Abstract

Fragile X Syndrome (FXS) is the most common inherited cause of intellectual disability and autism. It results from expansion of a CGG nucleotide repeat in the 5′ untranslated region (UTR) of FMR1. Large expansions elicit repeat and promoter hyper-methylation, heterochromatin formation, FMR1 transcriptional silencing and loss of the Fragile X protein, FMRP. Efforts aimed at correcting the sequelae resultant from FMRP loss have thus far proven insufficient, perhaps because of FMRP’s pleiotropic functions. As the repeats do not disrupt the FMRP coding sequence, reactivation of endogenous FMR1 gene expression could correct the proximal event in FXS pathogenesis. Here we utilize the Clustered Regularly Interspaced Palindromic Repeats/deficient CRISPR associated protein 9 (CRISPR/dCas9) system to selectively re-activate transcription from the silenced FMR1 locus. Fusion of the transcriptional activator VP192 to dCas9 robustly enhances FMR1 transcription and increases FMRP levels when targeted directly to the CGG repeat in human cells. Using a previously uncharacterized FXS human embryonic stem cell (hESC) line which acquires transcriptional silencing with serial passaging, we achieved locus-specific transcriptional re-activation of FMR1 messenger RNA (mRNA) expression despite promoter and repeat methylation. However, these changes at the transcript level were not coupled with a significant elevation in FMRP protein expression in FXS cells. These studies demonstrate that directing a transcriptional activator to CGG repeats is sufficient to selectively reactivate FMR1 mRNA expression in Fragile X patient stem cells.

On the wrong DNA track: Molecular mechanisms of repeat-mediated genome instability

“…Typically, potential therapies aim at the downstream effects arising from repeat expansion to reduce their toxic consequences. The goal is to either reverse gene silencing (70,81,469,(503)(504)(505) or alleviate adverse effects of toxic RNA (98, 101, 203, 506 -514) and/or toxic proteins (98,101,203,506,515). These methods, while worthy and promising, warrant several caveats.…”

Antisense oligonucleotide therapy rescues aggresome formation in a novel Spinocerebellar Ataxia type 3 human embryonic stem cell line

“…Donation and derivation of NIH-approved, unaffected hESC line UM4-6 (Registration # -0147, referred to here as WT-hESC) was reported previously22 . The SCA3-affected embryo was donated to the University of Michigan following single gene preimplantation genetic testing identified the embryo as heterozygous for a pathogenic CAG-repeat expansion in ATXN3.…”

Cell Reprogramming With CRISPR/Cas9 Based Transcriptional Regulation Systems

“…In further studies, different oligomers of VP16 (VP48, VP160, and VP192) have been used as activators ( Cheng et al, 2013 ; Duellman et al, 2017 ; Haenfler et al, 2018 ). It seems obvious that the efficiency of activation should grow with multiplexing of the VP16 domain and, indeed, VP160 usually demonstrates greater efficiency than VP64, but actually there is no convincing evidence that this correlation is true for all VP(16)n activators in all circumstances; rather it depends much more on the biological context.…”