Downregulation of PMP22 ameliorates myelin defects in iPSC-derived human organoid cultures of CMT1A

Abstract

Charcot-Marie-Tooth (CMT) disease is the most common inherited disorder of the peripheral nervous system. CMT1A accounts for 40-50% of all cases and is caused by a duplication of the PMP22 gene on chromosome 17, leading to dysmyelination in the peripheral nervous system. Patient-derived models to study such myelination defects are lacking as the in vitro generation of human myelinating Schwann cells has proven to be particularly challenging. Here, we present an iPSC-derived organoid culture, containing various cell types of the peripheral nervous system, including myelinating human Schwann cells, which mimics the human peripheral nervous system. Single-cell analysis confirmed the peripheral nervous system-like cellular composition and provides insight into the developmental trajectory. We used this organoid-model to study disease signatures of CMT1A, revealing early ultrastructural myelin alterations, including increased myelin periodic line distance and hypermyelination of small axons. Furthermore, we observed the presence of onion bulb-like formations in a later developmental stage. These hallmarks were not present in the for CMT1A-corrected isogenic line or in a CMT2A iPSC line, supporting the notion that these alterations are specific to CMT1A. Downregulation of PMP22 expression using short-hairpin RNAs or a combinatorial drug consisting of baclofen, naltrexone hydrochloride and D-sorbitol, was able to ameliorate the myelin defects in CMT1A-organoids. In summary, this self-organizing organoid model is able to capture biologically meaningful features of the disease and capture the physiological complexity, forms an excellent model to study demyelinating diseases, and supports the therapeutic approach of reducing PMP22 expression.

Protein expression profiles in brain organoids are more similar to those in human brain parenchyma than in mouse brain parenchyma

“…Fair et al (2020) detected spontaneous burst activity every 25 seconds, whereas Sharf et al (2022) observed activity every five seconds and every 125 seconds in Popova et al (2021). BOs generated using alternative protocols exhibited a slow firing rate in line with the results by Popova et al (2021, with burst activity being detected every 80 seconds (Van Lent et al, 2023). The electrophysiology parameters of our BOs were similar to Fair et al (2020) and Sharf et al (2022), as we detected burst activity approximately every 12 seconds.…”

Charcot–Marie–Toothneuropathies: Current gene therapy advances and the route toward translation

“…Lentiviral vectors were the first to be tested as vehicles to carry coding DNA (cDNA) or microRNA (miR) to treat models of CMT neuropathies, including CMT1X, [23][24][25][26] CMT4C 27 and CMT1A. 28,29 Despite the encouraging results shown in these studies and the large transgene capacity ($8 kb), lentiviruses resulted in relatively low transgene expression. Along with their inherent ability to integrate into the host genome, potentially leading to insertional mutagenesis, 30 lentiviruses are limited as a vehicle for in vivo clinical application.…”

“…shRNAs targeting the 3 0 UTR of human PMP22 were also cloned into a lentiviral system, and their efficiency was tested in vitro using a selforganizing iPSC-derived CMT1A organoid culture. 29 This culture is characterized by PMP22 overexpression and presents the key characteristics of the myelin pathology in CMT1A patients. shRNA treated CMT1A organoids silenced PMP22 and reversed hypermyelination, as…”

“…Lentiviral vectors were the first to be tested as vehicles to carry coding DNA (cDNA) or microRNA (miR) to treat models of CMT neuropathies, including CMT1X, 23–26 CMT4C 27 and CMT1A 28,29 . Despite the encouraging results shown in these studies and the large transgene capacity (~8 kb), lentiviruses resulted in relatively low transgene expression.…”

“…A single AAV2/9‐shRNA injection resulted in reduced Pmp22 levels, normalization of skin biomarker levels, improved myelination, and prevention of motor and sensory defects for up to 12 months post injection. shRNAs targeting the 3′UTR of human PMP22 were also cloned into a lentiviral system, and their efficiency was tested in vitro using a self‐organizing iPSC‐derived CMT1A organoid culture 29 . This culture is characterized by PMP22 overexpression and presents the key characteristics of the myelin pathology in CMT1A patients.…”