CRISPR/Cas9 assisted stem cell therapy in Parkinson's disease

Abstract

Since its discovery in 2012, CRISPR Cas9 has been tried as a direct treatment approach to correct the causative gene mutation and establish animal models in neurodegenerative disorders. Since no strategy developed until now could completely cure Parkinson's disease (PD), neuroscientists aspire to use gene editing technology, especially CRISPR/Cas9, to induce a permanent correction in genetic PD patients expressing mutated genes. Over the years, our understanding of stem cell biology has improved. Scientists have developed personalized cell therapy using CRISPR/Cas9 to edit embryonic and patient-derived stem cells ex-vivo. This review details the importance of CRISPR/Cas9-based stem cell therapy in Parkinson's disease in developing PD disease models and developing therapeutic strategies after elucidating the possible pathophysiological mechanisms.

Keywords: Disease model; Embryonic stem cells; Gene editing; Human pluripotent stem cells; Neurodegenerative disorder; α-synuclein.

Fig. 1

Overview of the pathogenesis of Parkinson's Disease. Various risk factors are involved in the pathogenesis of PD. Inherited as well as acquired gene mutations result in the generation of abnormal proteins. In the neuron, SNCA gene mutations encode an abnormal α-synuclein protein which forms aggregates and accumulates due to abnormal proteostasis mechanisms (UPS and ALP). Mutations in PINK1, PRKN, DJ-1, LRRK2, and PGC-1α are associated with mitochondrial dysfunction, resulting in ATP depletion, oxidative stress, and activation of neuroinflammatory pathways. Environmental chemicals like rotenone directly inhibit mitochondrial complex 1. The resulting DAMPs protein (damage-associated molecular patterns) and other pathologic events like K + efflux and lysosomal damage stimulate the NLRP3 inflammasome activation in the microglia, resulting in the generation of pro-inflammatory cytokines. These cytokines further propagate the inflammation, creating a vicious cycle that ultimately leads to the neuron's demise. DA: Dopaminergic, ALP: Autophagy-lysosomal pathway; UPS: Ubiquitin proteasome pathway; TLR: Toll-like receptor; ROS: Reactive oxygen species

Fig. 2

Mechanisms of CRISPR/Cas9 based gene editing. A. Schematic representation of mechanism of CRISPR/Cas9 based gene editing. Cas9 scans the DNA for the specific PAM sequence with the help of PAM interacting (PI) domain. Once the PAM sequence is identified sgRNA melts the target nucleotides upstream to PAM sequence and pairs them with crRNA. Then Cas9 cuts the target DNA 3 base pairs upstream of PAM sequence. Double strand breaks are repaired by Non homologous end joining (NHEJ) (majority) and Homology directed repair (HDR) resulting in random and precise deletions and insertions respectively B. CRISPR Cas9 based transcriptional regulation. Dead Cas9 (dCas9) is fused with specific transcription regulatory domains. Upon binding to the target DNA site, dCas9- regulatory domain complex recruits the corresponding gene activators/repressors to carry out the regulatory function C. CRISPR/Cas9 based base editing. dCas9 or Cas9 nickase (nCas9) are used for base editing. Cytosine base editors deaminate cytosine to uracil which is recognised by the DNA repair mechanisms and is substituted with Adenosine, whereas Adenosine base editors deaminate Adenosine to inosine which is substituted with Guanine by repair mechanisms

Fig. 3

Timeline for application of CRISPR/Cas9 tool in PD research

Fig. 4

Applications of CRISPR Cas9 tool in Parkinson's Disease. A. In-vitro application of CRISPR/Cas9 tool. Plasmids, Cas9 mRNA, SgRNA, or whole Ribonucleoprotein can be delivered into the biological systems using various methods. In-vitro applications in PD include the identification of pathogenic genes, model development, and the development of therapeutic strategies. B. In vivo applications of the CRISPR/Cas9 tool includes PD model development and screening of therapeutic strategies