Stem Cell Models and Gene Targeting for Human Motor Neuron Diseases

Abstract

Motor neurons are large projection neurons classified into upper and lower motor neurons responsible for controlling the movement of muscles. Degeneration of motor neurons results in progressive muscle weakness, which underlies several debilitating neurological disorders including amyotrophic lateral sclerosis (ALS), hereditary spastic paraplegias (HSP), and spinal muscular atrophy (SMA). With the development of induced pluripotent stem cell (iPSC) technology, human iPSCs can be derived from patients and further differentiated into motor neurons. Motor neuron disease models can also be generated by genetically modifying human pluripotent stem cells. The efficiency of gene targeting in human cells had been very low, but is greatly improved with recent gene editing technologies such as zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALEN), and CRISPR-Cas9. The combination of human stem cell-based models and gene editing tools provides unique paradigms to dissect pathogenic mechanisms and to explore therapeutics for these devastating diseases. Owing to the critical role of several genes in the etiology of motor neuron diseases, targeted gene therapies have been developed, including antisense oligonucleotides, viral-based gene delivery, and in situ gene editing. This review summarizes recent advancements in these areas and discusses future challenges toward the development of transformative medicines for motor neuron diseases.

Keywords: amyotrophic lateral sclerosis; antisense oligonucleotides; axonopathy; gene editing; gene therapy; human pluripotent stem cells; motor neuron diseases; neurodegeneration; spinal muscular atrophy; viral vectors.

Figure 1

Schematic representation of the gene editing tools. (a) zinc-finger nucleases (ZFNs) have Zn Finger domain that recognizes and binds the DNA strand and is fused with the Fok I nuclease. The Fok I nuclease requires dimerization in the opposite orientation to nick the DNA. (b) TALENs comprise the TALE DNA binding domain fused with the nuclease. The amino acids at certain positions called repeated variable di-residues (RVDs) recognize one of three nucleotides in the DNA strand to nick the DNA. (c) CRISPR Cas9 system with the sgRNA that recognizes specific target sites. The crRNA binds and destabilizes the DNA double helix, while the tracrRNA recruits the Cas9 protein which recognizes the PAM site and nicks the target DNA sequence.

Figure 2

Generation of induced pluripotent stem cells (iPSCs) and their applications. The adult somatic cells are reprogrammed to form the iPSCs using the pluripotency factors. These iPSCs can be directly differentiated to cell types from the three germ layers and used for disease modeling, drug screening and cell therapy. Alternatively, the iPSCs can be edited by gene editing technologies for gene insertion, gene disruption, and gene correction. These gene edited iPSCs can then be used for further applications.