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

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 require^2,3.

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 GWAS^5,6.

One of the key models used to study IBD pathogenesis^7,8 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 models^{10,11,12,13,14}. 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.