High-fidelity CRISPR/Cas9- based gene-specific hydroxymethylation rescues gene expression and attenuates renal fibrosis

Elisabeth M Zeisberg,Melanie S Hulshoff,Xingbo Xu,Tobias Moser,Xiaoying Tan,Michael Zeisberg,Gerd Hasenfuss,Björn Tampe,Tim Wilhelmi,Raghu Kalluri,Shoji Saito

doi:10.1038/s41467-018-05766-5

Abstract

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. TET enzymes catalyze hydroxymethylation of methylated DNA, reactivating gene expression. We here report generation of a high-fidelity CRISPR/Cas9-based gene-specific dioxygenase by fusing an endonuclease deactivated high-fidelity Cas9 (dHFCas9) to TET3 catalytic domain (TET3CD), targeted to specific genes by guiding RNAs (sgRNA). We demonstrate use of this technology in four different anti-fibrotic genes in different cell types in vitro, among them RASAL1 and Klotho, both hypermethylated in kidney fibrosis. Furthermore, in vivo lentiviral delivery of the Rasal1-targeted fusion protein to interstitial cells and of the Klotho-targeted fusion protein to tubular epithelial cells each results in specific gene reactivation and attenuation of fibrosis, providing gene-specific demethylating technology in a disease model.

Highlights

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy
We previously demonstrated that (1) TET3 is the predominant ten-eleven translocation (TET) protein in the kidney[5], (2) kidney fibrosis is associated with decreased TET3 expression[5], and (3) induction of endogenous TET3 expression leads to hydroxymethylation, demethylation, and thereby reactivation of various genes, including RASAL1, within diseased kidneys and attenuates experimental kidney fibrosis[5,14]
We demonstrate gene-specific targeting and successful re-expression of hypermethylated genes RASAL1, EYA1, LRFN2, and KLOTHO through all-in-one constructs in which either dCas[9] or high-fidelity dCas[9], respectively, is fused to the TET3 catalytic domain which is targeted to the promoters of these genes by single-guide RNA

Summary

Introduction

While suppression of specific genes through aberrant promoter methylation contributes to different diseases including organ fibrosis, gene-specific reactivation technology is not yet available for therapy. Reversal of hypermethylated Klotho promoter associated Klotho suppression by a lipophilic anthraquinone compound, Rhein, has been demonstrated to ameliorate renal fibrosis in unilateral ureter obstruction (UUO)-induced fibrotic kidney mouse model This results through effectively reducing aberrant DNMT1/3a expression and thereby maintaining secreted and membrane Klotho levels[22]. While nucleotide analogs are in clinical use in several malignant diseases such as myelodysplastic syndrome as demethylating therapies, they are highly unspecific and their utility is limited to second line therapies due to side effects, highlighting the need for gene-specific, less toxic demethylating therapies In this regard, members of the ten-eleven translocation (TET) family of zinc finger proteins (ZFPs) catalyze oxidation of methylated cytosine residues (so-called hydroxymethylation), which subsequently leads to replacement of methylated cytosine residues with naked cytosine[23]. There are more than 9000 genes targeted by TET proteins within the human genome, suggesting gene-specific delivery of TET as an attractive approach to rescue expression of aberrantly methylated genes[24]

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