Introduction: The ability to reprogram adult somatic cells into induced pluripotent stem cells (iPSCs) and the subsequent development of protocols for their differentiation into disease-relevant cell types have enabled in-depth molecular analyses of multiple disease states as hitherto impossible. Parkinson’s disease (PD) is one such example, in which the dopaminergic neurons that manifest pathology are embedded in an inaccessible region of the midbrain, the substantia nigra pars compacta, making it unfeasible to obtain samples of the affected region from living patients. Overabundance of alpha-synuclein (SNCA) has long been implicated in the pathogenesis of PD. However, the precise mechanism by which overexpression of SNCA leads to the demise of dopaminergic neurons remains elusive. Neurons differentiated from PD patient-specific iPSCs carrying multiplications of the SNCA gene may thus provide a means to recapitulate molecular phenotypes of the disease in vitro. The application of CRISPR/Cas9 to mammalian systems is likewise revolutionizing the utilization of genome editing in the study of molecular contributors to pathogenesis of numerous diseases, including PD.
Methods: We applied the nuclease-null or “dead” Cas9 (dCas9), fused to transcriptional activators and repressors to exert precise control over gene expression, in the absence of permanent alterations to the genome.
Results: Here, we describe the use of the transcriptional repressor, dCas9-KRAB, to silence SNCA gene expression in PD patient-specific iPSC-derived neurons harboring a triplication of the SNCA gene locus. In parallel, we have demonstrated that the transcriptional activator, dCas9-VPR, can be used to activate endogenous SNCA expression in iPSC-derived neurons from a healthy control patient to levels that parallel SNCA overexpression due to triplication.
Conclusions: The ability to exert precise transcriptional control over disease-associated gene expression in iPSC-derived neurons using dCas9 will further aid efforts to dissect molecular contributors to PD and many other neurodegenerative diseases. In addition, this method can be further developed to perform precise alterations of gene expression, or “genome surgery”, without generating permanent alterations to the genome, rendering this a safer platform for therapeutic development.
Patient Care: The implications of this research in patient care are two-fold: 1) combining the dCas9 system with patient-specific iPSC-derived neurons to investigate the earliest molecular pathogenic events that result from commonly implicated gene expression changes (i.e. amyloid precursor protein, Huntingtin, MAP-tau, alpha-synuclein) towards identifying early-acting preventative therapies, 2) developing this system as a safe and efficacious therapeutic platform for “genome surgery” in the brain.
Learning Objectives: By the conclusion of this session, participants should be able to 1) describe the limitless principle of coupling of transcriptional activators and repressors to an RNA-guided DNA binding protein scaffold for investigational use, 2) understand the advantages of using nuclease-null or “dead” Cas9 for making reversible gene expression changes in human neurons, 2) think critically about possible future therapeutic uses of this system for conducting “genome surgery” in the brain