Lentiviral Vector Platform for the Efficient Delivery of Epigenome-editing Tools into Human Induced Pluripotent Stem Cell-derived Disease Models

Abstract

The use of hiPSC-derived cells represents a valuable approach to study human neurodegenerative diseases. Here, we describe an optimized protocol for the differentiation of hiPSCs derived from a patient with the triplication of the alpha-synuclein gene (SNCA) locus into Parkinson's disease (PD)-relevant dopaminergic neuronal populations. Accumulating evidence has shown that high levels of SNCA are causative for the development of PD. Recognizing the unmet need to establish novel therapeutic approaches for PD, especially those targeting the regulation of SNCA expression, we recently developed a CRISPR/dCas9-DNA-methylation-based system to epigenetically modulate SNCA transcription by enriching methylation levels at the SNCA intron 1 regulatory region. To deliver the system, consisting of a dead (deactivated) version of Cas9 (dCas9) fused with the catalytic domain of the DNA methyltransferase enzyme 3A (DNMT3A), a lentiviral vector is used. This system is applied to cells with the triplication of the SNCA locus and reduces the SNCA-mRNA and protein levels by about 30% through the targeted DNA methylation of SNCA intron 1. The fine-tuned downregulation of the SNCA levels rescues disease-related cellular phenotypes. In the current protocol, we aim to describe a step-by-step procedure for differentiating hiPSCs into neural progenitor cells (NPCs) and the establishment and validation of pyrosequencing assays for the evaluation of the methylation profile in the SNCA intron 1. To outline in more detail the lentivirus-CRISPR/dCas9 system used in these experiments, this protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome-and genome-editing applications using hiPSCs and NPCs. The protocol

An Improved Protocol for the Production of Lentiviral Vectors

“…Functional titers will vary depending on the expression cassette packaged. It was found that using this protocol, the functional viral titer for Sp1-LVs carrying CRISPR/Cas9 transgenes was around 1 × 1,010 TU/mL per ∼5 × 10 7 producer cells ( Vijayraghavan and Kantor, 2017 ) and ( Tagliafierro et al., 2019 ).…”

Lentiviral Vectors for Delivery of Gene-Editing Systems Based on CRISPR/Cas: Current State and Perspectives

“…We demonstrated that cells targeted with LV-CRISPR/Cas9-DNMT3A showed substantial improvement in PD phenotypes, including nuclear transport, nuclear shape and integrity of the nuclear envelope, resistance to various stress stimuli, and others [ 65 ]. The optimized protocol for production of a lentivirus-CRISPR/dCas9 system at high-titers for transduction into primary cells, including hiPSCs and NPCs used in the above experiments, was published in [ 81 ]. The protocol describes how to produce, purify, and concentrate lentiviral vectors and to highlight their suitability for epigenome- and genome-editing applications.…”

Delivery Platforms for CRISPR/Cas9 Genome Editing of Glial Cells in the Central Nervous System

“…A unique LV-based CRISPR/Cas9 system has recently been generated to simultaneously deliver the Cas9 nuclease and four different sgRNAs, each under the control of a different promoter, thus allowing the simultaneous editing of different cell types in targeted tissues (Kabadi et al, 2014 ). Additionally, an all-in-one LV carrying dCas9 fused with the catalytic domain of DNA-methyltransferase 3A (DNMT3A) has recently been tested to target SNCA triplication in hiPSC-derived dopaminergic neurons to efficiently reduce SNCA expression levels, rescuing mitochondrial ROS production and cellular viability (Kantor et al, 2018 ; Tagliafierro et al, 2019 ). Importantly, expression cassettes driven by astrocyte-specific [e.g., gfaABC(1)D and gfaABC(1)D(B3)] and oligodendrocyte-specific [e.g., myelin basic protein ( MBP ) and 2,3-cyclic nucleotide 3-phosphodiesterase ( CNP )] promoters readily fit within LV genome, favoring glia-specific expression of editing enzymes by using an “all-in-one” system (McIver et al, 2005 ; Kagiava et al, 2014 ; Merienne et al, 2017 ; Humbel et al, 2020 ).…”