In Vitro CRISPR/Cas9-Directed Gene Editing to Model LRRK2 G2019S Parkinson’s Disease in Common Marmosets

Abstract

Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common mutation, associated with 1-3% of Parkinson's disease (PD) cases worldwide. G2019S is hypothesized to increase LRRK2 kinase activity. Dopaminergic neurons derived from induced pluripotent stem cells of PD patients carrying LRRK2 G2019S are reported to have several phenotypes compared to wild type controls, including increased activated caspase-3 and reactive oxygen species (ROS), autophagy dysfunction, and simplification of neurites. The common marmoset is envisioned as a candidate nonhuman primate species for comprehensive modeling of genetic mutations. Here, we report our successful use of CRISPR/Cas9 with repair template-mediated homology directed repair to introduce the LRRK2 G2019S mutation, as well as a truncation of the LRRK2 kinase domain, into marmoset embryonic and induced pluripotent stem cells. We found that, similar to humans, marmoset LRRK2 G2019S resulted in elevated kinase activity. Phenotypic evaluation after dopaminergic differentiation demonstrated LRRK2 G2019S-mediated increased intracellular ROS, decreased neuronal viability, and reduced neurite complexity. Importantly, these phenotypes were not observed in clones with LRRK2 truncation. These results demonstrate the feasibility of inducing monogenic mutations in common marmosets and support the use of this species for generating a novel genetic-based model of PD that expresses physiological levels of LRRK2 G2019S.Leucine-rich repeat kinase 2 (LRRK2) G2019S is a relatively common cause of Parkinson's disease (PD) 1 . Located in the kinase domain of LRRK2, G2019S is hypothesized to slow transition of the kinase from active to inactive forms, thus effectively increasing Vmax of the enzyme by ~2 fold. This increased kinase activity has been identified as the basis of many PD-associated dysregulated intracellular mechanisms 2-7 . Relative to other common PD-associated gene mutations (e.g. SNCA, Parkin, PINK1, GBA), LRRK2 G2019S has a relatively high penetrance 1,8,9 . The prevalence, penetrance, and sequence conservation of this mutation makes it an excellent target for genetic modeling of PD in a nonhuman species.Because of its genetic similarity to the human genome and shorter lifespan compared to rhesus monkeys 10 , the common marmoset (Callithrix jacchus) has emerged as a candidate nonhuman primate species for modeling age-related disorders, including PD. Genetic approaches for modeling PD (or other diseases) in monkeys has been achieved by introducing the mutant gene using viral vector technologies, via direct intracerebral delivery or microinjection delivery in the oocyte 11,12 . In both cases, the mutant protein is over-expressed at supraphysiological levels. In addition, intracerebral delivery requires brain surgery, and its effects are limited to the target region, which contradicts the current understanding of neurodegeneration as a multisystem disease.Cj-iPSC parental wild type and LRRK2 G2019S clones were fixed and immunostained for the dopaminergic and neuronal markers...

Splice-Switching Antisense Oligonucleotides Reduce LRRK2 Kinase Activity in Human LRRK2 Transgenic Mice

“…In support of this hypothesis, a similar kinase-dead truncated LRRK2 protein, generated by frameshift mutations introduced into iPSCs by CRISPR-Cas9-directed gene editing, was recently shown to alter kinase-dependent cellular phenotypes. 30 Despite achieving up to 30% exon 41 skipping in some tissues treated with ASO 41-1 and nearly 100% skipping in human fibroblast cell lines, we did not observe a band corresponding to a truncated LRRK2 on our SDS gels using a polyclonal antibody recognizing both proteins. It is possible that the antibody is not sensitive enough to detect the truncated protein in the tissues, which is less abundant than the full-length protein.…”

“…A similar such kinase-dead isoform was recently shown to alter LRRK2 kinase-dependent cellular phenotypes. 30 In addition, LRRK2 has been reported to function as a dimer; 41 , 42 thus, if the exon 41-skipped LRRK2 mRNA is translated into a truncated protein, its interaction with an equal amount of full-length LRRK2 could create a non-functional dimer, resulting in a more dramatic cumulative reducing effect on LRRK2 kinase activity through this dominant negative activity. Normalizing the pathologically elevated LRRK2 kinase activity to more physiological levels while minimally affecting LRRK2 protein levels may contribute to conservation of other non-kinase-related LRRK2 protein functions such as nuclear envelope integrity.…”

Melatonin and Autophagy in Aging-Related Neurodegenerative Diseases

“…In autosomal dominant PD, SNCA (encodes α-synuclein), LRRK2 (encodes leucine repeat kinase 2 involved in autophagy and other processes), VPS35 (encodes endosomes and vacuolar protein 35 in vesicles), DNAJC13 (encodes REM-8), and CHCHD2 (encodes mitochondrial protein) were mutated. Among them, LRRK2 mutation was the most common cause of delayed-onset PD [ 112 , 139 ]. In autosomal recessive PD, genes for Parkin (E3 ubiquitin ligase), PINK1 (PTEN-induced kinase 1), and DJ-1 (Park7, an antioxidant defensive enzyme and deglycosylase) are mutated and related to the occurrence of early-onset PD, and Parkin mutations are more frequent.…”

Induced Pluripotent Stem Cells from Animal Models: Applications on Translational Research

“…These cells were mainly generated from fibroblasts and integrative methods, but more recently, they were produced through non-integrative methods, such as Sendai-virus and episomal vectors [10,[35][36][37]. NH-primate-derived iPSCs have been used in research related to or as models for neurological [38][39][40][41], cardiac [36,42,43], reproductive [44], hematopoietic conditions [37,45], transplantation and grafting [30,46] and others.…”

“…More recently, ZFNs and TALENs have been superseded by CRISPR/Cas9, which is equally, if not more efficient in inducing double-strand breaks (DSBs) and in stimulating homologydirected repair (HDR) [53,54], also offering improved target specificity, prediction of off-target effects and activity [53,55,56]. Those approaches have been successfully applied to generate various NH-primate models (Reviewed by [57,58]), including the above mentioned Huntington's disease transgenic monkey [59], Parkinson's [41,60], neurodevelopmental disorders [61], Duchenne muscular dystrophy [62], severe combined immunodeficiency [63], and others.…”