Functional CRISPR repair of induced pluripotent stem cells from patients with Noonan syndrome-associated cardiac hypertrophy

by Lennart Roos née Lennart Roos
Doctoral thesis
Date of Examination:2024-05-21
Date of issue:2024-05-17
Advisor:Prof. Dr. Gerd Hasenfuß
Referee:Prof. Dr. Gerd Hasenfuß
Referee:Prof. Dr. Bernd Wollnik
Referee:Prof. Dr. Ralf Dressel
Persistent Address: http://dx.doi.org/10.53846/goediss-10501

 

 

Files in this item

Name:Roos_Lennart_Dissertation_eDiss.pdf
Size:3.95Mb
Format:PDF

The following license files are associated with this item:

Abstract

English

Noonan syndrome (NS) is a multisystemic disorder and characterised by its vast clinical variety including craniofacial dysmorphism, short stature and congenital heart defects resulting in severe pulmonary stenosis, hypertrophic cardiomyopathy (HCM), and other cardiac anomalies. With an estimated prevalence of one in 1000 to 2500 live births and a common autosomal-dominant inheritance, NS is the second most frequent congenital heart disease after trisomy 21. Caused by mutations encoding the RAS/MAPK signaling pathway and its components, NS is classified as a RASopathy (Roberts et al. 2013). Exploration of biallelic autosomal-recessive loss-of-function mutations within Leucine Zipper Like Transcription Regulator 1 (LZTR1) in cardiomyocytes derived from induced pluripotent stem cell (iPSC-CMs) allowed identification of the underlying pathomechanism causing severe-onset NS-associated HCM (Hanses et al. 2020). LZTR1 is known to attenuate RAS activity through ubiquitination and is hypothesised to act as a ‘RAS-killer’, inhibiting RAS-hyperactivity, with cellular processes disinhibited upon loss-of-function (Abe et al. 2020). In-depth molecular and functional studies of patient-specific iPSC-CMs recapitulated the hypertrophic disease phenotype showing severely elevated RAS/MAPK signaling levels and impaired calcium handling (Hanses et al. 2020). This project was able to correct, reverse and thus repair the disease-causing deep-intronic mutation c.1943-256C>T in LZTR1 in patient-specific iPSCs utilizing CRISPR/Cas9 as a precise genome editing tool. The intronic editing approach chosen to disrupt the disease-causative additional donor splice site, thereby preventing installation of a cryptic exon, restored the expression of functional LZTR1. Furthermore, the isogenic-CRISPR-controls confirmed the families’ mutations as disease-causing. The extensive in vitro exploration of the CRISPR-corrected-iPSC-CMs demonstrated a reversal of hypertrophy by normalised cell size compared to hypertrophic NS-iPSC-CMs, as well as attenuation of RAS protein to WT levels (Hanses et al. 2020). By combining molecular approaches with structural and functional investigations, this study ultimately led to a better understanding of the underlying disease pathomechanisms of this severe phenotype of NS. This improved understanding may pave the way for more effective diagnostic and therapeutic approaches in patient-specific precision medicine. In conclusion, this study presented an efficient, safe, and clinically translatable CRISPR-based gene therapy targeting a deep-intronic mutation in LZTR1 to cure the NS-associated pathology in a preclinical iPSC-CM screening platform. Translation of this CRISPR-based strategy could lead to the development of a promising therapeutic approach for the clinic (Knauer et al. 2024).
Keywords: Noonan syndrome; RASopathy; RAS-MAPK pathway; LZTR1; Hypertrophic cardiomyopathy; Disease modeling; iPSCs; iPSC-CMs; gene therapy; genome editing; CRISPR-Cas9; stem cells
 
Statistik