Replacing murine insulin 1 with human insulin protects NOD mice from diabetes

Abstract

2 16 Abstract 17 Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-18 producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop 19 autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been 20 the preeminent murine model for human type 1 diabetes research for several decades. However, 21 humanized mouse models are highly sought after because they offer both the experimental 22 tractability of a mouse model and the clinical relevance of human-based research. Autoimmune 23 T-cell responses against insulin, and its precursor proinsulin, play central roles in the 24 autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first 25 step towards developing a murine model of the human autoimmune response against pancreatic 26 beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene 27 (INS) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin 28 in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-29 peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild 30 type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15-20% of HuPI 31 mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice.32 Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days 33 but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 34 to create mouse models of human diseases that express proteins pivotal to the human disease.35 Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes. 36 3 37 Introduction 38 Type 1 diabetes (T1D) is an autoimmune disease caused by the T-cell mediated destruction of 39 the pancreatic, insulin-producing beta cells [1, 2]. This leads to a primary insulin deficiency and 40 subsequent metabolic disease [3]. Since its discovery in 1980 [4], the Non-obese diabetic (NOD) 41 mouse has been the preferred mouse model for research into human T1D. Autoimmune diabetes 42 in the NOD mouse shares many pathological features with human type 1 diabetes [5]. For 43 example, diabetes develops spontaneously, the MHC II genes greatly impact upon susceptibility 44 and immune-cell infiltration of the islets of Langerhans is observed [6]. 45 46 Many autoantigens have been implicated in the immune pathogenesis of human T1D and NOD 47 autoimmune diabetes [7, 8]. However, T cells specific for insulin, and its precursor proinsulin, 48 have been shown to cause diabetes in the NOD mouse [9-11]. Although pathogenesis cannot be 49 measured directly in humans, T-cell responses to (pro)insulin are strongly implicated in human 50 type 1 diabetes [12-15]. Humans carry one insulin gene (INS), whereas the mouse genome 51 contains two nonallelic insulin genes, Ins1 (chromosome 19) and Ins2 (chromosome 7) [16]. Ins152 is predomin...

Beta-cell Cre expression and reduced Ins1 gene dosage protect mice from type 1 diabetes

“…Our findings concur with previous work that showed a similar reduction in diabetes incidence in female NOD mice lacking 1 or 2 alleles of Ins1 (10,15), although we did not observe additional protection with the double Ins1 knockout, beyond what was seen with the Cre replacement allele. Similarly, our data are in alignment with a previous study showing replacing the murine Ins1 gene with the human INS gene was found to protect female NOD mice from diabetes in both heterozygous and homozygous states (17). We were unable to detect differences in diabetes incidence in male Ins1 Cre/WT , Ins1 Neo/WT , and Ins1 Neo/Cre NOD mice, perhaps due to an insufficient study period.…”

“…Insulin is a primary auto-antigen in both murine and human type 1 diabetes pathogenesis (1). Replacing Ins1 with non-antigenic human insulin also protects NOD mice from onset of diabetes (17). Together, these previous observations indicate that insulin 1 gene dosage is a key driver of diabetes in NOD mice.…”

Insulin’s other life: an autoantigen in type 1 diabetes

“…replaced murine Ins1 with the human insulin gene. This genetic modification almost completely protected NOD mice from developing diabetes, 44 indicating that human insulin, which is more similar to murine insulin 2, is not able to completely replace murine Ins1 as a target of autoimmune responses. Because the expression of human insulin, in this knock‐in model, is driven by the native murine Ins1 promoter, it is unlikely that this phenotype is attributable to changes in expression of inulin in the thymus.…”

“…[41][42][43] Using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPRassociated protein 9), Elso et al replaced murine Ins1 with the human insulin gene. This genetic modification almost completely protected NOD mice from developing diabetes, 44 indicating that human insulin, which is more similar to murine insulin 2, is not able to completely replace murine Ins1 as a target of autoimmune responses.…”

Sex Differences in the Development of Hyperglycemia in Rodent Models of Diabetes and Obesity

“…While rodent models represent many similarities of human diabetes mellitus, there are limitations for direct clinical use. Considering the insulin gene, humans possess a single copy of the gene on chromosome 11, whereas mice carry two genes, Ins1 and Ins2 [86]. Additionally, mouse models have key variations in physiology, anatomy, and psychology that create challenges for direct comparisons of diabetes mechanisms and treatments in humans [87].…”