Summary

Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies

Published: June 02, 2021
doi:

Summary

Here, we describe the methodology to knock out a gene of interest in the immune system using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and the evaluation of these mice in a cluster of differentiation 40 (CD40) agonistic antibody-induced colitis model.

Abstract

The immune system functions to defend humans against foreign invaders such as bacteria and viruses. However, disorders of the immune system may lead to autoimmunity, inflammatory disease, and cancer. The inflammatory bowel diseases (IBD)-Crohn’s disease (CD) and ulcerative colitis (UC)-are chronic diseases marked by relapsing intestinal inflammation. Although IBD is most prevalent in Western countries (1 in 1,000), incident rates are increasing around the world. Through association studies, researchers have linked hundreds of genes to the pathology of IBD. However, the elaborate pathology behind IBD and the high number of potential genes pose significant challenges in finding the best therapeutic targets. Additionally, the tools needed to functionally characterize each genetic association introduce many rate-limiting factors such as the generation of genetically modified mice for each gene. To investigate the therapeutic potential of target genes, a model system has been developed using clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated endonuclease (Cas9)-based technologies and a cluster of differentiation 40 (CD40) agonistic antibody. The present study shows that CRISPR/Cas9-mediated editing in the immune system can be used to investigate the impact of genes in vivo. Limited to the hematopoietic compartment, this approach reliably edits the resulting reconstituted immune system. CRISPR/Cas9-edited mice are generated faster and are far less expensive than traditional genetically modified mice. Furthermore, CRISPR/Cas9 editing of mice has significant scientific advantages compared to generating and breeding genetically modified mice such as the ability to evaluate targets that are embryonic lethal. Using CD40 as a model target in the CD40 agonistic antibody-induced colitis model, this study demonstrates the feasibility of this approach.

Introduction

Autoimmune diseases refer to conditions in which a patient's immune system attacks their own cells and organs, resulting in chronic inflammation and tissue damage. Nearly 100 different types of autoimmune conditions have been described to date, affecting 3-5% of the human population1. Many of the autoimmune conditions, including systemic lupus erythematosus and IBD, lack effective treatments and present significant unmet medical needs. Currently affecting around 1.5 million people in the USA alone, IBD is a devastating disease marked by progressive, persistent, and relapsing intestinal inflammation with no available cure. Unraveling the underlying pathogenesis and pathophysiology is needed to deliver the novel treatment and prevention strategies that IBD patients require2,3.

Over 230 different IBD loci have been identified through genome-wide association analyses (GWAS)4. Although these associations have elucidated new genes that are potentially important players in the key mechanisms and pathways of IBD, only a few genes from these loci have been studied. Some genes have been implicated in specific pathways. For example, the microbe-sensing pathway has been linked to nucleotide-binding oligomerization domain-containing protein 2 (NOD2); the autophagy pathway has been linked to autophagy-related 16 like 1 (ATG16L1), immunity-related GTPase family M (IRGM), and caspase recruitment domain family member 9 (CARD9); and the pro-inflammatory pathway has been linked to interleukin (IL)-23-driven T-cell responses4. Various in vivo mouse models have been used to functionally characterize genes identified through GWAS5,6.

One of the key models used to study IBD pathogenesis7,8 is the CD40 model of colitis, which induces innate immune intestinal inflammation following the injection of a CD40 agonistic antibody into immunodeficient (T and B-cell) mice. Primarily used to examine the contribution of innate immunity to IBD development, mostly macrophages and dendritic cells9, it is unclear if disease can be induced in fully immune-competent wild-type (WT) mice. In addition to animal models, gene-specific tools are also required for the functional characterization of a gene, including chemical compounds and biologics. More importantly, genetically modified animals are essential in revealing the function of a specific gene. However, the strategies typically used to make genetically modified mice-embryo injection and breeding-often take over a year and incur a significant financial cost. This rate-limiting process presents a significant challenge in the quest to elucidate the functions of the IBD-related genes identified by GWAS.

The protocol presented here provides a viable alternative to breeding genetically modified mice. First, as shown in the Figure 1 schematic, lineage-negative, stem cell antigen1-positive, receptor tyrosine kinase Kit-positive (lineage-Sca1+c-Kit+ or LSK) cells are isolated from the bone marrow of Cas9 knockin (KI) mice bearing a specific allele (CD45.2) to allow donor immune cell tracking. Next, these cells are exposed to lentiviruses bearing different guide RNAs (gRNAs) and a fluorescent marker, violet-excited green fluorescent protein (VexGFP), to allow tracking of transduced cells. Two days later, VexGFP+ cells are sorted and injected into lethally irradiated recipient Ly5.1 Pep Boy mice, which are C57Bl/6 mice bearing the CD45.1 allele to allow for recipient immune cell tracking. Twelve weeks later, the immune system is fully reconstituted, and the mice can be enrolled into in vivo models.

In addition to the benefit of cost savings and faster time-to-generation compared to the generation and breeding of genetically modified animals, this methodology is ideal for targets that are embryonic lethal, as it specifically targets the hematopoietic compartment. Furthermore, for targets where there are no tools available, such as an antibody, this system provides a feasible approach. In summary, to address the challenges described thus far, an in vivo CRISPR/Cas9-based genome editing platform was developed to expeditiously generate genetically modified animal models10,11,12,13,14. This study demonstrates that intestinal inflammation in WT C57Bl/6 mice can be induced by a CD40 agonistic antibody. CD40 is a key regulator of disease in this model and was therefore used as a model target to validate the CRISPR/Cas9-based knockout and loss of gene function.

Protocol

All animal experiments performed following this protocol must be approved by the respective Institutional Animal Care and Use Committee (IACUC). All procedures described here were approved by the AbbVie IACUC. 1. Generation of required lentiviruses and procurement of donor and recipient animals NOTE: The Table of Materials includes source and order number details for all animals, instruments, and reagents used in this protocol. Construct…

Representative Results

Following the procedure described above, mice expressing CD40-targeted gRNA were generated. By week 2, B-cells, CD11b+ macrophages, and CD11c+ dendritic cells (DCs) were engrafted (Figure 2). T-cells however, as expected based on previous literature18, took longer to fully engraft and required 12 weeks post-engraftment to reach ~90% (Figure 2). Immune organs, such as the spleen and lymph nodes, had the mos…

Discussion

The results shown here introduce a novel CRISPR/Cas9-based genome editing platform capable of investigating gene function in this CD40 agonistic antibody-induced colitis model. Cell sorting enriched the pool of genetically modified LSK cells, resulting in over 90% reduction in CD40 expression within the reconstituted animals-in just 4 months. Furthermore, the reduced expression of CD40 within the immune system had a profound effect within the CD40 agonistic antibody-induced colitis model, significantly reducing disease e…

Offenlegungen

The authors have nothing to disclose.

Acknowledgements

Thank you to Ruoqi Peng, Donna McCarthy, Jamie Erikson, Liz O'Connor, Robert Dunstan, Susan Westmoreland, and Tariq Ghayur for your efforts supporting this work. Thank you to Pharmacology leaders including Rajesh Kamath and others for their leadership in establishing the CD40 agonistic antibody-induced colitis model in WT C57Bl/6 mice. Additionally, thank you to all those at AbbVie Bioresearch Center and Cambridge Research Center in the Comparative Medicine East Department supporting in vivo experiments.

We would like to thank the Zhang lab from the Broad Institute and McGovern Institute of Brain Research at the Massachusetts Institute of Technology for providing CRISPR reagents [multiplex Genome Engineering Using CRISPR/Cas Systems. Cong, L, Ran, FA, Cox, D, Lin S, Barretto, R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F Science. 2013 Jan 3].

Materials

6-well tissue culture plates Corning/Costar #3506
TransIT-LT1 Mirus Bio MIR 2300/5/6
MACS Buffer (autoMACS Running Buffer) Miltenyi Biotec 130-091-221
0.45 µm filter unit Millipore #SLHV013SL
0.6 mL microcentrifuge Tube Axygen MCT-060-C-S
1.5 mL Eppendorf Tube Axygen MCT-150-C-S
15mL Conical VWR 21008-918
23 G Needle VWR #305145
24 Well Non-TC Plates Falcon #351147
24-Well TC Plates Falcon #353047
50 mL Conical tube VWR 21008-951
5 mL Syringe BD Biosciences #309647
70 µm Filter Miltenyi #130-098-462
96-Well Flat Bottom Plates Corning #3599
96-Well U-Bottom Plates Corning/Costar #3365
Anesthesia Machine VetEquip – COMPAC5 #901812
Anti-CD40 Agonist monoclonal antibody BioXcell BE-0016
Anti-p40 monoclonal antibody BioXcell BE-0051
B220 PE Antibody BioLegend #103208
Bovine serum albumin Sigma Aldrich A7906-100G
Cas9 Knock-in Mice Jackson Labs #026179 C57Bl/6 background
CD117+ Beads Miltenyi #130-091-224
CD11b PE Antibody BioLegend #101208
CD3 PE Antibody BD Biosciences #553240
Centrifuge Beckman Coulter Allegra 6KR Centrifuge
Countertop Centrifuge Eppendorf Centrifuge 5424
DPBS ThermoFisher #14190136
Dulbecco’s Modified Eagle Medium Mediatech #10-013-CV
Ethylenediamine tetraacetic acid (EDTA) Invitrogen AM9260G
Endoscope Karl Storz N/A Custom Coloview Tower
Flow cytometer BD Biosciences FACS Aria II
Fms-related tyrosine kinase 3 ligand (Flt-L) PeproTech #250-31L
Gr-1 PE Antibody BD Biosciences #553128
Hank's balanced salt solution (HBSS) ThermoFisher #14170120
Heat-Inactivated Fetal Bovine Serum HyClone #SH30071.03
IL-7 PeproTech #217-17
Incubator Binder #9040-0116
Isoflurane HenrySchein #6679401710
LS Column Miltenyi #130-042-041
Ly5.1 Pepboy Mice Jackson Labs #002014 C57Bl/6 background
mouse stem cell factor (mSCF) PeproTech #250-03
Sodium chloride (NaCl) Hospira #00409488850
OPTI-MEM serum-free media Invitrogen #31985-070
Penicillin-streptomycin (PenStrep) ThermoFisher #15140-122
Plate Shaker ThermoFisher #88880023
pLentiPuro Addgene #52963
Polybrene (10 µg/µL) Sigma Aldrich #TR-1003-G
Red Blood Cell Lysis Buffer eBioscience #00-4333
Retronectin Takarbio #T100B
Sca-1 APC Antibody BioLegend #108112
StemSpan StemCell Technologies #09600
Ter119 PE Antibody eBioscience #12-5921
Thrombopoietin (TPO) PeproTech #315-14
X-ray Irradiator Precision X-Ray X-Rad 320

Referenzen

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Graham, S., Gao, L., Wang, R. Investigating Target Gene Function in a CD40 Agonistic Antibody-induced Colitis Model using CRISPR/Cas9-based Technologies. J. Vis. Exp. (172), e61618, doi:10.3791/61618 (2021).

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