Minimally Humanized Exon-18 Mouse Cell Lines Validate Preclinical CRISPR/Cas9 Approach to Treat Weaver Syndrome.

Weaver syndrome is a rare neurodevelopmental disorder that encompasses macrocephaly, tall stature, obesity, brain anomalies, intellectual disability, and increased susceptibility to cancer. This dominant monogenic disorder is caused by germline variants in enhancer of zeste 2 polycomb repressive complex 2 subunit (), a key epigenetic writer. Unfortunately, there are no effective treatments for Weaver syndrome. However, preclinical results support the potential for therapeutic gains, despite the prenatal onset. Thus, for the first time, we tested whether CRISPR/Cas9 gene-editing strategies may be able to "correct" a Weaver syndrome variant at the DNA level. We initiated these preclinical studies by humanizing the region surrounding the most-common recurring patient-variant location in mouse embryonic stem cells (ESCs). Humanization ensures that DNA-binding strategies will be directly translatable to human cells and patients. We then introduced into ESCs the humanized region, but now carrying the Weaver syndrome variant c.2035C>T p.Arg684Cys, and characterized the enzymatic properties of this missense variant. Our data showed a significant and dramatic reduction in EZH2-enzymatic activity, supporting previous cell-free studies of this variant as well as and mouse work by other teams. Intriguingly, this most-common variant does not create a complete loss-of-function, but rather is a hypomorphic allele. Together with prior reports describing hypomorphic effects of missense variants, these results demonstrate that the etiology of Weaver syndrome does not require complete loss of EZH2 enzymatic activity. Toward therapy, we tested four CRISPR gene-editing strategies. We demonstrated that Cas9 (Cas9) showed the highest variant correction (70.5%), but unfortunately also the highest alteration of the nonvariant allele (21.1-26.2%), an important consideration for gene-editing treatment of a dominant syndrome. However, Cas9 (Cas9) gave a variant correction (52.5%) that was not significantly different than Cas9, and encouragingly the lowest alteration of the nonvariant allele (2.0%). Thus, the therapeutic strategy using the small Cas9 enzyme, a size that allows flexibility in therapeutic delivery, was the most optimal for targeting the Weaver syndrome variant c.2035C>T p.Arg684Cys.