From gene therapy to somatic mutagenesis : insights into the common Hutchinson-Gilford progeria syndrome mutation

Abstract

Mutations of the human genome can cause a large variety of genetic diseases, such as the Hutchinson-Gilford progeria syndrome (HGPS). The LMNA c.1824C>T point mutation amplifies a cryptic splice event within exon 11 of the LMNA gene, producing a toxic protein called progerin. Progerin accumulation leads to a large variety of severe clinical manifestations, such as premature atherosclerosis, with a reduced median lifespan of 14,6 years for affected patients. With the development of next-generation gene editing tools such as base editors, these pathogenic single nucleotide variants (SNVs) can be corrected. Furthermore, Next-generation sequencing technology has identified somatic mutations in many human tissues. Therefore, we investigated the HGPS LMNA c.1824C>T point mutation as a somatic mutation in chronic kidney disease (CKD) patients based on an early vascular aging phenotype commonly observed in CKD and HGPS patients.

In paper I, we examined the effects of progerin expression on the symmetry of stem cell division within the skin. Here, we observed a shift in skin cell division symmetry based on progerin expression due to an impaired Wnt signaling pathway and the mislocalization of nuclear envelope proteins. This shift towards symmetrical stem cell division likely promotes a stem cell exhaustion phenotype, inducing aberrant tissue homeostasis. In paper II, we investigated a gene therapy approach using an adenine base editor (ABE) to correct the LMNA c.1824C>T HGPS point mutation in the skin of HGPS mice, previously characterized in study I. Delivery of the ABE-VQR and the point mutation targeting sgRNA to skin cells with a transient non-integrative lentiviral particle vector system resulted in an initial mutation correction of 20,8-24,1%. Long-term analysis revealed an improvement of the HGPS skin phenotype, likely mediated by a corrected skin stem cell population. In paper III, we studied the occurrence of the LMNA c.1824C>T point mutation as a somatic mutation in CKD patients due to early vascular aging phenotype similarities with HGPS patients. We obtained CKD patient arteries of living-donor kidney transplantations and observed progerin-expressing vascular smooth muscle cells (VSMCs). These progerinexpressing VSMCs appeared in solitary and clusters, suggesting clonal expansion. Furthermore, we observed the LMNA c.1824C>T point mutation in 78,3% of DNA samples from CKD patient arteries, where some samples displayed a high fractional mutation abundance of up to 49,7%. Furthermore, we could detect the LMNA c.1824C>T mutation in peripheral blood samples from all CKD patients, with an allelic mutation fraction of 0,012- 0,047%.

In conclusion, we investigated a shift in stem cell division symmetry in the skin of HGPS mice due to Wnt signaling pathway changes that potentially contribute to an aberrant tissue homeostasis phenotype. We explored a novel gene therapy approach, using an ABE to correct the LMNA c.1824C>T point mutation, which was transiently delivered in the skin of HGPS mice. ABE-treated HGPS mice displayed long-term improvements of the skin phenotype, likely mediated by a corrected skin stem cell population. Furthermore, we investigated the LMNA c.1824C>T point mutation as a somatic mutation in CKD patient arteries. We observed progerin expressing VSMCs in solitary and cluster and detected the HGPS point mutation in the majority of CKD patients, even at a high fractional abundance.