258. Universal Stem Cell Gene Therapy Platform: Broadening the Transient Epigenetic Gene Therapy Arm by Using Nuclease-Null dCas9 as an Additional Drive for Efficient Epigenetic Editing
Authors: Roger Bertolotti
Journal: Molecular Therapy (2015)

Bacterial CRISPR/Cas9 is an RNA-guided endonuclease that has been converted into a breakthrough tool for human gene targeting culminating in efficient gene repair/genomic editing and is now emerging, through its nuclease-null dCas9 double-mutant, as a popular new drive for human endogenous gene control culminating in epigenomic editing. Cas9 overwhelming strength is its easy and inexpensive customization to DNA targets since it relies on a short single-guided RNA. Therefore, dCas9 has already been successfully used as a programmable RNA-guided DNA-binding module for efficient targeting of dimeric FokI endonuclease and a powerful tripartite transcriptional activator. Fusing dCas9 to a variety of epigenetic effector domains stands thus as an attractive option to broaden the transient epigenetic gene therapy arm of our proposed 2005 Universal Stem Cell Gene Therapy Platform. Coined as a translational application of human promoter-specific siRNA-mediated transcriptional gene silencing (promoter methylation) and promoter demethylation mediated by non-coding antisense-RNA, transient epigenetic gene therapy is aimed at long-term transcriptional gene silencing/activation through the transient action of promoter/enhancer-specific siRNAs/dsRNAs or short sense/antisense RNAs/oligonucleotides (oligos). Unlike canonical RNA interference mediated by siRNAs that target cytoplasmic mRNAs for post-transcriptional gene silencing through sequence-specific mRNA cleavage or translational repression, promoter-specific small RNAs/oligos-mediated transcriptional gene silencing/activation is a long-lasting epigenetic process that involves DNA methylation/demethylation and post-translational histone modifications. Therefore, transient epigenetic gene therapy should provide a new dimension to basic gene therapeutics in which the transient action of a dCas9-Methylase/Demethylase fusion protein or of promoter/enhancer-specific small RNAs/oligos could be used for long-term up-/down-regulation of target genes. Transient epigenetic gene therapy has exciting applications for inherited diseases such as allele-specific silencing for autosomal-dominant mutants or activation of fetal genes as substitutes for mutant adult cognates. However, long-term transcriptional up-/down-regulation of target genes has many other applications since promoter/enhancer-specific small RNAs/oligos target non-coding sense/antisense RNAs including long ones, i.e. the very RNAs that appear to drive the complex digital regulation of human epigenetic processes and inherent pathologies. Therefore, our approach is discussed in terms of new therapeutic/prophylactic avenues for 1) epigenetic pathologies such as neurodevelopmental disorders originating from deleterious early life epigenetic programming (eg depression, schizophrenia), 2) therapeutic remodeling of imprinted gene expression (eg Angelman syndrome), 3) degenerative diseases and age-related disorders (eg Alzheimer's & Parkinson's diseases), 4) cancer and infectious diseases, 5) the ex vivo genesis of clinical-grade iPS cells and 6) rising stem cell conversion.