CRISPR/CAS9 AND PIGGYBAC MEDIATED LRRK2-G2019S IN VITRO PARKINSON’S DISEASE MODELING

Parkinson’s disease (PD), the second most common neurodegenerative disorder, is characterized by the progressive loss of dopaminergic (DA) neurons in the substantial nigra pars compacta (SNpc) area of the human midbrain with an unclear cause. Mutations revealed by whole genome sequencing (WGS) from familial PD cases may explain how cell loss occurs. Confirmation of this hypothesis has been hampered by the lack of available cell types from affected patients. Transgenic animal models have been used, but differences between these animals and humans have greatly impacted their usefulness for studying human diseases. Additionally, because PD is regarded to only affect humans, reliable human material-based experimental models are urgently needed. Human-induced pluripotent stem cells (hiPSCs)- derived DA neurons provide an opportunity to establish in vitro mutation-related PD models of disease-relevant cells that represent replacement alternatives to in vivo animal experiments. However, these hiPSCs-based PD models have limitations regarding the genetic background differences between patients and healthy controls. Genomic editing of hiPSCs allows for the generation of isogenic cellular models that differ only in the disease-specific mutations of interest. Currently, the biggest concern regarding nuclease-mediated genomic editing is the potentially undesirable alterations associated with remnant sequences, off-target effects and random integration, which may result in cell lines not being truly isogenic. To avoid potential confounding effects and establish a causal link between genotype and phenotype, robust isogenic cell lines free of unwanted mutagenesis are absolutely required for the study of PD. To better understand the pathogenesis of the most prevalent leucine-rich repeat kinase 2 (LRRK2) mutation, G2019S, which causes both familial and sporadic PD, patient hiPSCs have been corrected using the Cre/LoxP recombination system. However, the LoxP site inevitably remaining after excision of the selection cassette can influence gene expression. In this thesis, a “footprint-free” LRRK2-G2019S isogenic model was created using clustered regularly interspaced short palindromic repeats/Cas9-associated (CRISPR/Cas9) system and a piggyBac transposon that can remove selection cassettes without leaving remnants. In LRRK2-G2019S DA neurons, the percentage of tyrosine hydroxylase (TH)-positive neurons with a total neurite length greater than 2 cm was significantly reduced, and the average branch number was also decreased. These PD-like phenotypes could be rescued by administration of the specific LRRK2 inhibitor LRRK2-IN-1 and by the compound BRF110, which activates the Nurr1:RXRa heterodimer to replenish the DA shortage. Our data suggest that the “footprint- free” LRRK2-G2019S isogenic cell lines allow standardized, genetic background-independent, in vitro PD modeling and are suitable for screening novel drugs that have clinical applications. In addition, we have shown that in vitro TH-positive neurons with a total neurite length greater than 2 cm were positive for serine 129 phosphorylated (S129P) α-synuclein, and we hypothesize that S129P α-synuclein plays a role in the maintenance or formation of long neurites. Thus, we have also provided new insights into the roles of LRRK2-G2019S and S129P α-synuclein in PD pathogenesis. Furthermore, we have optimized CRISPR/Cas9-mediated genomic editing in hiPSCs by establishing a FACS-assisted CRISPR/Cas9 editing (FACE) strategy that uses three fluorescent proteins to isolate biallelic-edited cells with no random integration and by using Exonuclease III (ExoIII)-facilitated long single stranded DNA (ssDNA) donor to reduce random integration