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

Summary

Targeted DNA epigenome editing represents a powerful therapeutic approach. This protocol describes the production, purification, and concentration of all-in-one lentiviral vectors harboring the CRISPR-dCas9-DNMT3A transgene for epigenome-editing applications in human induced pluripotent stem cell (hiPSC)-derived neurons.

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 is easily adaptable and can be used to produce high titer lentiviruses for in vitro and in vivo applications.

Introduction

Multiple epigenome-editing platforms have been recently developed to target any DNA sequences in the regions that control gene expression^1,2. The created epigenome-editing tools are designed to (i) regulate transcription, (ii) alter posttranslational histone modifications, (iii) modify DNA methylation, and (iv) modulate regulatory element interactions. The approach to anchor the transcription/chromatin modifiers to a deactivated (dead) Cas9 (dCas9) raised from previously developed epigenome-editing platforms, such as zinc finger proteins (ZFPs) and transcription activator-like effectors (TALEs), harboring a po....

Protocol

1. System Design and Virus Production

1. Plasmid design and construction
   NOTE: The construction of an all-in-one LV-gRNA-dCas9-DNMT3A vector is performed by using a production-and expression-optimized expression cassette published by Ortinski et al.³⁰. The vector cassette carries a repeat of the recognition site of transcription factor Sp1 and a state-of-the-art deletion within the untranslated (U3') region of a 3'-long terminal repeat (LTR.......

Discussion

LVs have begun to emerge as the vehicle of choice for epigenome editing, especially in the context of genetic diseases, mainly due to their ability to (i) accommodate large DNA payloads and (ii) efficiently transduce a wide range of dividing and nondividing cells. The large packaging efficacy of the LVs is especially beneficial for the applications involving packaging of the CRISPR/dCas9 systems which are oversized. From this perspective, LVs represent the platform-of-choice for the delivery of all-in-one CRISPR/Cas9 sys.......

Materials

Name	Company	Catalog Number	Comments
Equipment
Optima XPN-80 Ultracentrifuge	Beckman Coulter	A99839
0.22 μM filter unit, 1L	Corning	430513
0.45-μm filter unit, 500mL	Corning	430773
100mm TC-Treated Culture Dish	Corning	430167
15 mL conical centrifuge tubes	Corning	430791
150 mm TC-Treated Cell Culture dishes with 20 mm Grid	Corning	353025
50mL conical centrifuge tubes	Corning	430291
6-well plates	Corning	3516
Aggrewell 800	StemCell Technologies	34811
Allegra 25R tabletop centrifuge	Beckman Coulter	369434
BD FACS	Becton Dickinson	338960
Conical bottom ultracentrifugation tubes	Seton Scientific	5067
Conical tube adapters	Seton Scientific	PN 4230
Eppendorf Cell Imaging Slides	Eppendorf	30742060
High-binding 96-well plates	Corning	3366
Inverted fluorescence microscope	Leica	DM IRB2
QIAprep Spin Miniprep Kit (50)	Qiagen	27104
Reversible Strainer	StemCell Technologies	27215
SW32Ti rotor	Beckman Coulter	369650
VWR® Disposable Serological Pipets, Glass, Nonpyrogenic	VWR	93000-694
VWR® Vacuum Filtration Systems	VWR	89220-694
xMark™ Microplate Absorbance plate reader	Bio-Rad	1681150
Name	Company	Catalog Number	Comments
Cell culture reagents
Human embryonic kidney 293T (HEK 293T) cells	ATCC	CRL-3216
Accutase	StemCell Technologies	7920
Anti-Adherence Rinsing Solution	StemCell Technologies	7010
Anti-FOXA2 Antibody	Abcam	Ab60721
Anti-Nestin Antibody	Abcam	Ab18102
Antibiotic-antimycotic solution, 100X	Sigma Aldrich	A5955-100ML
B-27 Supplement (50X), minus vitamin A	Thermo Fisher Scientific	12587010
BES	Sigma Aldrich	B9879 - BES
Bovine Albumin Fraction V (7.5% solution)	Thermo Fisher Scientific	15260037
CHIR99021	StemCell Technologies	72052
Corning Matrigel hESC-Qualified Matrix	Corning	08-774-552
Cosmic Calf Serum	Hyclone	SH30087.04
DMEM-F12	Lonza	12-719
DMEM, high glucose media	Gibco	11965
DNeasy Blood & Tissue Kit	Qiagen	69504
EpiTect PCR Control DNA Set	Qiagen	596945
EZ DNA Methylation Kit	Zymo Research	D5001
Gelatin	Sigma Aldrich	G1800-100G
Gentamicin	Thermo Fisher Scientific	15750078
Gentle Cell Dissociation Reagent	stemCell Technologies	7174
GlutaMAX	Thermo Fisher Scientific	35050061
Human Recombinant bFGF	StemCell Technologies	78003
Human Recombinant EGF	StemCell Technologies	78006
Human Recombinant Shh (C24II)	StemCell Technologies	78065
MEM Non-Essential Amino Acids Solution (100X)	Thermo Fisher Scientific	11140050
mTeSR1	StemCell Technologies	85850
N-2 Supplement (100X)	Thermo Fisher Scientific	17502001
Neurobasal Medium	Thermo Fisher Scientific	21103049
Non-Essential Amino Acid (NEAA)	Hyclone	SH30087.04
PyroMark PCR Kit	Qiagen	978703
RPMI 1640 media	Thermo Fisher Scientific	11875-085
SB431542	StemCell Technologies	72232
Sodium pyruvate	Sigma Aldrich	S8636-100ML
STEMdiff Neural Induction Medium	StemCell Technologies	5835
STEMdiff Neural Progenitor Freezing Medium	StemCell Technologies	5838
TaqMan Assay FOXA2	Thermo Fisher Scientific	Hs00232764
TaqMan Assay GAPDH	Thermo Fisher Scientific	Hs99999905
TaqMan Assay Nestin	Thermo Fisher Scientific	Hs04187831
TaqMan Assay OCT4	Thermo Fisher Scientific	Hs04260367
TaqMan Assay PPIA	Thermo Fisher Scientific	Hs99999904
Trypsin-EDTA 0.05%	Gibco	25300054
Y27632	StemCell Technologies	72302
Name	Company	Catalog Number	Comments
p24 ELISA reagents
Monoclonal anti-p24 antibody	NIH AIDS Research and Reference Reagent Program	3537
Goat anti-rabbit horseradish peroxidase IgG	Sigma Aldrich	12-348	Working concentration 1:1500
Goat serum, Sterile, 10mL	Sigma	G9023	Working concentration 1:1000
HIV-1 standards	NIH AIDS Research and Reference Reagent Program	SP968F
Normal mouse serum, Sterile, 500mL	Equitech-Bio	SM30-0500
Polyclonal rabbit anti-p24 antibody	NIH AIDS Research and Reference Reagent Program	SP451T
TMB peroxidase substrate	KPL	5120-0076	Working concentration 1:10,000
Name	Company	Catalog Number	Comments
Plasmids
pMD2.G	Addgene	12253
pRSV-Rev	Addgene	52961
psPAX2	Addgene	12259
Name	Company	Catalog Number	Comments
Restriction enzymes
BsmBI	New England Biolabs	R0580S
BsrGI	New England Biolabs	R0575S
EcoRV	New England Biolabs	R0195S
KpnI	New England Biolabs	R0142S
PacI	New England Biolabs	R0547S
SphI	New England Biolabs	R0182S