Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening.

Konrad Schwefel,Christiane D Much,Oliver Otto,Ute Felbor,Uwe Völker,Stefanie Spiegler,Sabine Ameling,Robin A Pilz,Matthias Rath

doi:10.1111/jcmm.14075

Abstract

CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.

Highlights

Cerebral cavernous malformations (CCM) are clusters of thin‐walled, sinusoidal vascular channels within the brain or spinal cord
Using the non‐viral and selection‐ free CRISPR/Cas[9] RNP delivery approach, we observed CCM3 indel frequencies in human endothelial cells (ECs) that were significantly higher than those described for other target genes so far.[28]
With NGS‐based tracking of CCM3 mutant allele frequencies after genome editing and use of a limiting dilution cloning assay, we have clearly shown that CCM3–/– ECs have a clonal survival advantage

Summary

| INTRODUCTION

Cerebral cavernous malformations (CCM) are clusters of thin‐walled, sinusoidal vascular channels within the brain or spinal cord. Little is known about the long‐term effects of CCM3 inactivation due to homozygous or compound heterozygous loss‐of‐function CCM3 variants in human ECs. CCM3 was initially described as an. Upregulated transcript in human TF‐1 erythroleukaemia cells upon GM‐CSF deprivation and was reported as CCM disease gene in 2005.7,8 Pull‐down and co‐immunoprecipitation studies demonstrated that CCM3 forms a ternary complex with CCM1 and CCM2 in vitro and acts in intracellular networks with GCKIII serine/threonine kinases and other molecules.[9]. While the occurrence of potential off‐target mutations may limit the use of both techniques, the delivery of the CRISPR/Cas[9] components as ribonucleoprotein (RNP) complex can reduce off‐target effects due to its short intracellular half‐life.[27]. We used a crRNA:tracrRNA:Cas[9] RNP‐based model of human cavernous malformations and siRNA transfections in parallel to study the molecular and functional effects of long‐term CCM3 deficiency in comparison to its acute knockdown. We demonstrate that CCM3 inactivation in human ECs induces a clonogenic survival advantage of functionally impaired CCM3–/– ECs over time

| METHODS

| RESULTS

| DISCUSSION

Findings

CONFLICT OF INTEREST