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. 2019 Dec 10;14(12):e0225021. doi: 10.1371/journal.pone.0225021

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

Colleen M Elso 1,2, Nicholas A Scott 1,¤, Lina Mariana 1, Emma I Masterman 1, Andrew P R Sutherland 1,2, Helen E Thomas 1,2, Stuart I Mannering 1,2,*
Editor: Matthias G von Herrath3
PMCID: PMC6903741  PMID: 31821343

Abstract

Type 1, or autoimmune, diabetes is caused by the T-cell mediated destruction of the insulin-producing pancreatic beta cells. Non-obese diabetic (NOD) mice spontaneously develop autoimmune diabetes akin to human type 1 diabetes. For this reason, the NOD mouse has been the preeminent murine model for human type 1 diabetes research for several decades. However, humanized mouse models are highly sought after because they offer both the experimental tractability of a mouse model and the clinical relevance of human-based research. Autoimmune T-cell responses against insulin, and its precursor proinsulin, play central roles in the autoimmune responses against pancreatic beta cells in both humans and NOD mice. As a first step towards developing a murine model of the human autoimmune response against pancreatic beta cells we set out to replace the murine insulin 1 gene (Ins1) with the human insulin gene (Ins) using CRISPR/Cas9. Here we describe a NOD mouse strain that expresses human insulin in place of murine insulin 1, referred to as HuPI. HuPI mice express human insulin, and C-peptide, in their serum and pancreata and have normal glucose tolerance. Compared with wild type NOD mice, the incidence of diabetes is much lower in HuPI mice. Only 15–20% of HuPI mice developed diabetes after 300 days, compared to more than 60% of unmodified NOD mice. Immune-cell infiltration into the pancreatic islets of HuPI mice was not detectable at 100 days but was clearly evident by 300 days. This work highlights the feasibility of using CRISPR/Cas9 to create mouse models of human diseases that express proteins pivotal to the human disease. Furthermore, it reveals that even subtle changes in proinsulin protect NOD mice from diabetes.

Introduction

Type 1 diabetes (T1D) is an autoimmune disease caused by the T-cell mediated destruction of the pancreatic, insulin-producing beta cells [1, 2]. This leads to a primary insulin deficiency and subsequent metabolic disease [3]. Since its discovery in 1980 [4], the Non-obese diabetic (NOD) mouse has been the preferred mouse model for research into human T1D. Autoimmune diabetes in the NOD mouse shares many pathological features with human type 1 diabetes [5]. For example, diabetes develops spontaneously, the MHC II genes greatly impact upon susceptibility and immune-cell infiltration of the islets of Langerhans is observed [6].

Many autoantigens have been implicated in the immune pathogenesis of human T1D and NOD autoimmune diabetes [7, 8]. However, T cells specific for insulin, and its precursor proinsulin, have been shown to cause diabetes in the NOD mouse [9–11]. Although pathogenesis cannot be measured directly in humans, T-cell responses to (pro)insulin are strongly implicated in human type 1 diabetes [12–15]. Humans carry one insulin gene (INS), whereas the mouse genome contains two nonallelic insulin genes, Ins1 (chromosome 19) and Ins2 (chromosome 7) [16]. Ins1 is predominantly expressed in the beta cells and Ins2 is expressed in both the thymus and beta cells [17–19]. Genetic deletion of the Ins1 gene protects NOD mice from autoimmune diabetes [20], whereas Ins2 knock-out NOD mice rapidly develop autoimmune diabetes [21]. These observations suggest that insulin 2 protects NOD from diabetes, perhaps by promoting the deletion of (pro)insulin specific T cells in the thymus, whereas insulin 1 is primarily a target of diabetes-causing T cells.

The NOD mouse is a powerful research tool, but the relevance of findings from mouse models to human type 1 diabetes remain uncertain [22, 23]. Several groups have attempted to develop mouse models that harbor human cells, or express human genes, in an effort to develop models that are both experimentally tractable and clinically relevant. Broadly, these models fall into two camps: (i) immune-deficient mice that are transplanted with human cells [24, 25] and (ii) NOD mice that have been genetically manipulated to express relevant human genes [26, 27]. Human cell transplant models vary depending upon the donor cells and must be reestablished for each experiment. Genetic models are more defined, but the expression of human transgenes does not always follow that of the murine orthologues [28]. Variability of expression arises because the integration of the transgene is random, and expression is subject to the regulatory environment into which it integrates. Transgenes may also integrate in tandem arrays leading to variable expression levels depending upon the number of copies of the transgene. Furthermore, to avoid functional impacts of the endogenous murine genes these also need to be disrupted.

The emergence of CRISPR/Cas9 technology provides an opportunity to edit the mammalian genome with unprecedented precision [29]. Here we asked if CRISPR/Cas 9 could be used to replace the coding sequence of murine insulin with human insulin. This would allow the expression of human insulin, in place of murine insulin 1, with minimal disruption of the murine genome. A NOD mouse that expresses human insulin from the murine insulin 1 locus would be an important step towards developing a NOD mouse model that mimics the human T-cell response to proinsulin [12, 14]. Here we report the generation of a human insulin replacement mouse at the murine insulin 1 locus. We show that this mouse produces human insulin, develops insulitis, but is largely protected from diabetes, similarly to Ins1 knock-out NOD mice.

Materials and methods

Production of human insulin knock-in mice

All mouse experiments were approved by the Animal Ethics Committee of the St Vincent’s Hospital Melbourne (AEC:019.14 and 020.14) and carried out under the NHMRC Code of Practice. NOD/Lt-Ins1em1(INS)Tkay (HuPI) mice were created at the Australian Phenomics Network (APN, Melbourne, Australia) using CRISPR-Cas9-mediated replacement of murine Ins1 with human INS. sgRNAs were designed to target the 5’ and 3’ regions of Ins1. A homology-directed repair (HDR) template was produced by cloning into pUC19: 701bp 5’ homology arm, 333bp sequence encoding human INS and 506 bp 3’ homology arm (resulting in 559bp homology at 3’ end of the INS gene, see S1 Fig). NOD/Lt embryos were microinjected cytoplasmically with 30 ng/μl Cas9 mRNA, 15 ng/μl each sgRNA and 30 ng/μl linearized HDR template. In some experiments they were incubated in 50μM SCR7 (a non-homologous end joining (NHEJ) inhibitor [30]) overnight prior to transfer into recipients (S1 Table). The sequences and on- and off-target scores for the sgRNAs are shown (S1 Table). One of 51 pups contained the complete insert in the correct location (S2 Table). This pup arose from a microinjection using sgRNAs 1 and 3 (S1 Table). This was confirmed by PCR and sequencing. The mutation was backcrossed to NOD/Lt for at least two, but for almost all experimental mice, three, generations and intercrossed to produce human insulin knock-in homozygous mice. No differences were detected in mice from different numbers of backcrosses.

ELISAs for human insulin and C-peptide

Seven to twelve week old HuPI mice were fasted 5–6 h before collection of serum. Human insulin and C-peptide, and murine insulin were measured by ELISA (Mercodia, Uppsala, Sweden). Mouse insulin measurements were adjusted for cross-reactivity with human insulin as per manufacturer’s instructions. Data are expressed as mean ± SEM and differences assessed using an unpaired Student’s two-tailed t test.

PCR

RNA was extracted from mouse pancreas samples using Nucleospin RNA extraction columns (Macheray Nagal) after mechanical disruption. On column DNase digestion was performed as per manufacturer’s instructions. cDNA was made with Superscipt III (Invitrogen) using 400ng RNA and 2.5μM oligo-dT. Cloneamp HiFI Premix (Takara) was used to amplify cDNA with 0.2μM primers as per manufacturer’s instructions. PCR conditions: 35 cycles of 98°C 10 sec, annealing temp 15sec, 72°C 10sec, followed by 72°C 7min. Primer sequences and annealing temperatures can be found in S4 Table.

Glucose tolerance test

A glucose tolerance test was carried out on 6-10-week-old HuPI mice. Mice were fasted for 6 h before intraperitoneal injection of glucose solution (2g/kg, Baxter, Deerfield, IL, USA), followed by measurement of blood glucose at 0, 15, 30, 45, 60, and 120 min (Accu-Chek Performa, Roche). A two-way ANOVA was performed to detect differences between the groups.

Immunohistochemistry and immunofluorescence

Pancreata were fixed in 4% paraformaldehyde and embedded in paraffin. Pancreatic sections were stained with 4 μg/mL of anti-human proinsulin/C-peptide (GN-ID4, Developmental Studies Hybridoma Bank, IA, USA) or an isotype control (40BI3.2.1-s, Developmental Studies Hybridoma Bank, IA, USA) for 45 min at RT followed by rabbit anti-rat IgG-HRP (Dako) for 45 min at RT. Slides were then incubated for 1 min with 3,3’-Diaminobenzidine and counterstained with haematoxylin. No staining was seen in pancreas of wildtype NOD mice. For immunofluorescence, pancreata were frozen in OCT (optimal cutting temperature) compound, cryosectioned and fixed in acetone before staining with the anti-human proinsulin/C-peptide (GN-ID4) or isotype control (40BI3.2.1-s) antibodies, followed by staining with an anti-rat IgG-AF568.

Diabetes incidence

Age-matched HuPI homozygotes, heterozygotes and wildtype controls were tested weekly for diabetes by measuring their urinary glucose concentrations (Diastix, Bayer) until 300d of age. Mice were declared diabetic if they had high urinary glucose readings for three consecutive days and a blood glucose reading >15mmol/L (Accu-Check Proforma, Roche). Pairwise comparisons of the diabetes incidence between mouse strains were performed using the log-rank test.

Insulitis

Pancreata were collected from 100d old or 300d old mice, fixed in Bouin’s fixative and embedded in paraffin. Sections (5 μm) were cut at three different levels 100 μm apart and stained with haematoxylin and eosin. Lymphocytic infiltration of the islets was scored as described [31], briefly: 0 = no infiltration, 1 = peri-insulitis, 2 = < 25% islet infiltrated, 3 = > 25% of the islet infiltrated and 4 = complete infiltration. To calculate the insulitis score, the number of islets in each scoring category: 0, 1, 2, 3, 4, was multiplied by 0, 0.25, 0.5, 0.75 and 1, respectively. This value was then divided by the total number of islets to provide a weighted average insulitis score for each mouse. Differences between strains were assessed using a one-way ANOVA.

Results

Production of human insulin knock-in mice

Murine INS1 is predominantly expressed in the beta cell, whereas INS2 is expressed both in the thymus and beta cell [18, 19]. To ensure expression of human insulin in the beta cells of knock-in mice, we replaced the amino acid coding region of mouse Ins1 with the human INS coding sequence (Fig 1). Note that the amino acid sequence of the 16 amino acids at the COOH terminal end of insulin A-chain is identical between human and murine insulin, so the sequence of this region did not change (Fig 1). CRISPR-Cas9 was used to induce double strand DNA breaks close to the start and stop codons of Ins1 (S1 Table). The human coding sequence was introduced by homologous recombination from a repair construct containing homologous regions flanking the Ins1 coding region (Fig 1A and 1B). On initial screening, 5 of 51 founder mice contained the 3’ end of the human insulin gene (S2 Table). Further analysis confirmed that one founder mouse contained the entire human insulin coding sequence in the correct genomic location. This founder mouse was bred to WT NOD and was established as a line (called HuPI). These mice are healthy, viable and display no gross abnormalities. Offspring of an F1 intercross carried the knock-in allele at expected Mendelian ratios (S3 Table).

Fig 1. Replacement of the murine Ins1 gene with human INS.

Fig 1

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(A) Alignment of the amino acid sequence of murine and human insulin. Amino acids in murine insulin that differ from the human sequence are highlighted. (B) A schematic diagram of the replacement of the coding sequence for murine Ins1 with human INS. Murine sequences are shown in blue and boxes represent exons. The translation start and stop codons are indicated by arrows. A homology directed repair (HDR) construct containing the human INS transcript (purple box), flanked by sequence homologous to the regions flanking murine Ins1 was used to introduce INS by CRISPR-Cas9 mediated targeting to the Ins1 locus. (5’ homology: 701bp; 3’ homology: 559bp).

HuPI mice produce human insulin

The concentration of human insulin and C-peptide in HuPI serum was determined by ELISA (Fig 2A and 2B). Human insulin was detected in human insulin knock-in heterozygous mice, but higher concentrations of insulin (KI/+; 0.4–3.8 mU/L v KI/KI; 2.3–17.2 mU/L) and C-peptide (KI/+; 0–292 pmol/L v KI/KI; 28–556 pmol/L) were observed in human insulin homozygous knock-in mice. As expected, both human insulin and murine insulin could be detected in the serum from human insulin knock-in mice (Fig 2D). PCR analysis revealed that human insulin, but not murine insulin 1 encoding mRNA, could be detected in pancreatic extracts from HuPI mice (Fig 2C and S4 Table). In contrast, murine insulin 2 and beta actin mRNA were detected in pancreatic extracts from both WT and HuPI mice.

Fig 2. Human insulin and C-peptide can be detected in NOD.HuPI serum and NOD.HuPI mice have normal glucose tolerance.

Fig 2

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ELISA was used to measure (A) human insulin and (B) human C-peptide concentrations in serum from fasted NOD.HuPI wildtype (+/+, n = 7), heterozygous knock-in (KI/+, n = 10) and homozygous knock-in (KI/KI, n = 8) mice. Data are expressed as mean ± SEM. NS: p > 0.05, * p < 0.05, ** p < 0.01, **** p < 0.0001; unpaired Student’s two-tailed t test. (C) Detection of cDNA specific for human insulin (INS), murine insulin 1 (Ins1) and murine insulin 2 (Ins2) in pancreatic cells. (D) Human and mouse insulin were measured in serum from homozygous knock-in mice (n = 5). (E) A glucose tolerance test was performed on fasted female NOD.HuPI wildtype (+/+, n = 8), heterozygous knock-in (KI/+, n = 8) and homozygous knock-in (KI/KI, n = 8) mice. p > 0.05; two-way ANOVA.

Both human and murine insulin 2 are expressed in pancreatic extracts from HuPI mice. Comparison of murine and human insulin revealed that, although variable between mice, less human insulin was expressed compared to murine insulin (Fig 2D). Overall insulin production was similar; when more human insulin was expressed less murine insulin was produced. To evaluate the HuPI mice’s ability to metabolize glucose, a glucose tolerance test was performed. No difference in glucose tolerance was observed between NOD and HuPI mice (Fig 2E). To confirm that human insulin is expressed in pancreatic beta cell we analyzed insulin expression by immunohistochemistry. Human insulin was detected in beta cells from HuPI mice (Figs 3 and S2). We conclude that the HuPI mouse expresses murine insulin 2 and human insulin in place of mouse insulin 1.

Fig 3. Human insulin is localised to the islets of NOD.HuPI mice.

Fig 3

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Pancreas sections from human (A, E), NOD.HuPI wildtype (B, F), and homozygous knock-in (C, D, G, H) were stained with anti-human proinsulin (GN-ID4) (A–D, top row), or an isotype control antibody (40BI3.2.1-s) (E–H, bottom row). Positive staining is indicated by the brown color. No staining was seen in pancreas of HuPI wildtype mice. Photos taken at 100x magnification. Representative images are shown.

HuPI mice have a reduced incidence of diabetes

Human insulin knock-in mice, both homozygous and heterozygous, had a significantly reduced incidence of diabetes (p<0.0001, Log-rank test) compared to wildtype NOD mice when cohorts were followed for 300 days (Fig 4A). To assess the level of islet infiltration, pancreata were collected from HuPI homozygous, heterozygous and wildtype mice at approximately 100 days of age. At this time islet infiltration is commonly seen in wildtype NOD mice. Homozygous and heterozygous human insulin knock-in mice exhibited reduced insulitis at 100 days (Figs 4B and 4C and S3) compared to wildtype NOD mice. By 300 d, the insulitis in the HuPI heterozygous and homozygous was equal to wild type NOD mice (Figs 4B and 4C and S4).

Fig 4. NOD.HuPI mice have reduced incidence of diabetes and delayed insulitis.

Fig 4

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(A) Female homozygous NOD.HuPI knock-in (KI/KI, N = 30), heterozygous knock-in (KI/+, n = 27) or wildtype (+/+, n = 34) mice were aged for 300 days. Urinary glucose was tested weekly. Mice were declared diabetic if they had high urinary glucose readings for three consecutive days and a blood glucose reading >15mmol/L. **** p < 0.0001 Log-rank test +/+ vs KI/KI and +/+ vs KI/+. Lymphocytic infiltration of the islets was scored in haematoxylin and eosin-stained pancreas sections of female NOD.HuPI homozygous knock-in (KI/KI, n = 5), heterozygous knock-in (KI/+, n = 5) and wildtype (+/+, n = 3) 100d-old mice and female NOD.HuPI homozygous knock-in (KI/KI, n = 5), heterozygous knock-in (KI/+, n = 5) and wildtype (+/+, n = 4) 300d-old mice (B, C). Islet infiltration scoring: 0 = no infiltration, 1 = peri-insulitis, 2 = < 25% islet infiltrated, 3 = > 25% of the islet infiltrated and 4 = complete infiltration. A weighted average insulitis score was calculated as described in the methods. 100d p < 0.05; 300d p > 0.05; one-way ANOVA.

Discussion

Here we describe the generation and characterization of a NOD mouse that expresses human insulin in place of murine insulin 1. We show that human insulin is expressed in the pancreatic islets and human insulin and C-peptide is present in the serum. These mice breed normally, have normal glucose tolerance and are largely protected from diabetes.

Genetic modification by CRISPR/Cas9 raises the possibility that the phenotype observed is attributable to off-target genomic mutations. We believe that this is very unlikely for the following reasons. The founder lines were backcrossed to NOD for two, or in most cases, three generations. This reduces the load of putative off target mutations by >87%. No differences were noticed between mice in different branches of the pedigree, or with numbers of backcrosses. In addition, most controls were littermates of the mice harboring the HuPI insertion. The sgRNAs used for the CRISPR/Cas9 targeting resulting in the founder mutation had moderate off target scores of 47 and 67 (according to IDT’s algorithm: https://sg.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE) suggesting that they had a modest propensity to make off target modifications.

As expected, because the murine insulin 1 gene regulatory elements were left intact, human insulin expression was restricted to the islets of Langerhans. Human insulin and C-peptide were detected in the serum, suggesting that human proinsulin was processed to insulin and C-peptide by murine pancreatic beta cells. The HuPI mice showed no deficit in glucose tolerance, indicating that the combined levels of human insulin and murine insulin 2 were sufficient to maintain normal glucose regulation.

Surprisingly HuPI mice were protected from spontaneous diabetes. This is similar to the phenotype of murine Ins1 knock-out NOD mice [20]. Moriyama et al [20] found that approximately 10% more Ins1+/- mice developed diabetes than Ins1-/- mice at 300, whereas we did not see any difference in diabetes incidence between HuPI homozygous or heterozygous mice at this time (Fig 4A).

Currently it is not clear how human insulin protects NOD mice from diabetes. One possibility is that the NOD mouse repertoire is ‘blind’ to human insulin, despite the similarity in the protein sequence between human insulin and murine insulin 1 (Fig 1). However, this does not account for the protection from diabetes in the HuPI heterozygous mice. In the heterozygous mice the concentration of Ins1 may be below a critical threshold required for the onset of diabetes. It should be noted that HuPI mice were not completely protected from diabetes, but the incidence of disease was significantly reduced with a delayed onset. Histological analysis showed that there was little islet infiltration at 100 days, but this increased markedly by 300 days. Our incidence studies were stopped after 300 days of age however it is possible that the incidence of diabetes in the HuPI mice would increase if they were monitored for longer.

The amino acid sequences are identical between mouse and human proinsulin in the insulin B9-23 epitope (Fig 1A). NOD mice that carried a tyrosine (Y) to alanine (A) mutation position B16 are protected from diabetes [9]. Most of the amino acid differences between mouse and human proinsulin fall within the C-peptide. NOD mouse CD4 epitopes have been described from this region [32]. This suggests that changes in the sequence of proinsulin C-peptide may contribute to the low incidence of diabetes in HuPI mice. Recently we reported that full-length C-peptide is a major antigen in human type 1 diabetes [14]. Our current observations suggest it may also play a greater role in the NOD mouse than previously appreciated.

HuPI mice still express murine insulin 2. Human insulin was readily detected in the serum and islets of HuPI mice and mRNA for murine insulin 1 was undetectable. It will be of interest to generate NOD mice with both murine insulin 1 and insulin 2 replaced with human insulin, which would only make human insulin. Because proinsulin, the precursor of insulin, is the major protein product of beta cells, accounting for ~10% of the cell’s protein [33], it is possible that a fully human insulin knock-in mouse may have dysfunctional beta cells due to misfolding of human insulin in a murine beta cell. However, our current results suggest that a mouse that only expresses human insulin would be healthy. Murine insulin knockout mice that express human insulin from a transgene have been generated and appear to have no metabolic abnormalities [34].

To develop a murine model of human autoimmune responses that cause type 1 diabetes will require further modification of the HuPI mouse. Such a model will need to express T1D associated HLA alleles, namely HLA-DQ2, DQ8 (and their transdimers), HLA-DR4 or HLA-DR3 [35]. In addition, a TCR specific for human proinsulin, or other relevant beta-cell antigens, will be required. It will be a high priority to investigate the function of TCRs isolated from human islet infiltrating T cells [12], because these cells are most likely to be pathogenic [1]. The ‘Yes’ mouse is, to our knowledge, the most advanced transgenic model for human T1D [34]. This mouse expresses HLA-A2:01, HLA-DQ8 and carries human insulin as transgenes, with the murine orthologues of these genes knocked out. This model is on a mixed C57BL/6 and CBA background. One advantage of CRISPR/Cas9 mediated gene replacement is that it can be done on a NOD background, which avoids issues related to mixed genetics. Verhagen et al [27] recently reported an HLA-DR4 transgenic mouse that expresses CD80 on its pancreatic beta cells. Diabetes can be induced in this mouse by immunization with murine insulin 2 derived peptides in adjuvant. While these mice can be manipulated to develop diabetes, it is unclear how faithfully this model recapitulates the human disease.

At this stage it is unknown if human proinsulin specific TCRs, or T cells, will respond to human insulin expressed by the HuPI mice. Addressing this question will require a NOD mouse that expresses human CD4, (pro)insulin specific TCR and the restricting HLA allomorph for the T cell to be generated. Nonetheless, the feasibility of using CRISPR/Cas9 to replace murine genes with their human orthologues augers well for a new generation of mouse models for T1D and other human autoimmune diseases. Recent advances in using CRISPR/Cas9 to integrate large constructs into the murine genome will greatly assist in generating these models [36].

Supporting information

S1 Fig. Sequence of the homology-directed repair (HDR) template.

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S2 Fig. Human insulin is localized to the pancreatic islets in NOD.HuPI mice.

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S3 Fig. NOD.HuPI mice exhibit delayed insulitis (100d).

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S4 Fig. NOD.HuPI mice exhibit delayed insulitis (300d).

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S1 Table. Production of human insulin knock-in mice by CRISPR/Cas9 mutagenesis.

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S2 Table. Molecular confirmation of INS knock-in.

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S3 Table. Genotype ratios in offspring of NOD.HuPI mice.

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S4 Table. Insulin-specific primers.

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S1 Raw Image. Original gel image of Fig 2C.

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Acknowledgments

The mutant mice were produced via CRISPR/Cas9 oocyte injection by Monash University as a node of the Australian Phenomics Network (APN). The APN is supported by the Australian Government Department of Education through the National Collaborative Research Infrastructure Strategy, the Super Science Initiative and the Collaborative Research Infrastructure Scheme.

The authors thank the staff at St. Vincent’s Bioresources Centre for excellent animal husbandry.

Data Availability

All relevant data are within the manuscript and its Supporting Information files.

Funding Statement

This work was supported by a: A Millennium Award (Y17M1-MANS) from Diabetes Australia (SM) and a Diabetes Australia Project grant to CE (Y18G-ELSC); Australian National Health and Medical Research Council (GNT1123586) to SM and JDRF (JDRF 2-SRA-2018-568-S-B) to SM. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

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Decision Letter 0

Matthias G von Herrath

11 Nov 2019

PONE-D-19-29642

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

PLOS ONE

Dear A/Prof Mannering,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

==============================

ACADEMIC EDITOR: I like this paper  my main concern would be about off target effects /integrations ?  Matthias 

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Additional Editor Comments (if provided):

Overall I think this is a very interesting paper - my main concern would indeed relate to the issue whether there were any off targeted integrations? Disruptions? Matthias

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Reviewer #2: Yes

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Reviewer #1: This is an observational study where the authors have replaced the mouse INS1 gene with the human INS sequence and found that the resulting trangenic strain is protected against spontaneous autoimmune diabetes development. No mechnistic studies were performed so it is unclear why the mice are protected.

The key unknown in this story is whether the INS1/hINS replacement is indeed the (sole) cause of the reduced disease penetrance. CRISPR-Cas9 mediated double break induction and HDR is notoriously non-specific. Since no off-target analysis was performed at all, how certain are the authors that none of the breaks/insertions landed in e.g. an immune gene?

What do the authors mean by 'most of the amino acid coding region' being replaced (l142, p8). The Figure is too low resolution to check for myself.

The anitbodies used for ELISA and IHC to detect mouse vs human insulin: how certain are the authors that there is no cross-reactivity?

Reviewer #2: In this manuscript, entitled “Replacing murine insulin 1 with human insulin protects NOD mice from diabetes”, the authors describe the generation of a novel human insulin-expressing NOD mouse strain. To achieve this, the authors replaced the murine insulin 1 gene ( Ins1 ) in NOD mice with the human insulin gene ( Ins ) using Crispr/Cas9 technology. These mice (HuPI) express human insulin in place of murine insulin 1 in their islets, and human C-peptide can be detected in their serum. These mice show also lower incidence of T1D as compared to wild-type NOD mice. Undoubtedly, this can be a useful novel humanized model of T1D. Unfortunately, the study lacks immunological and overall mechanistic insights. Below we suggest a few, relatively simple experiments.

Comments/Suggestions

1. How do the authors explain the fact that KI/+ mice have the same incidence of T1D as KI/KI mice?

2. The authors need to discuss more the similarities and differences in the amino acid sequence between mouse ins1, ins2 and human insulin, particularly with regards the immunodominant epitopes, for example B:9-23. It would be of value to discuss Maki Nakayama’s paper in Nature 2004 within this context.

3. The authors need to measure mouse ins1 and 2 as well human insulin in the thymus of +/+, KI/+ and KI/KI mice.

4. Statistics are missing from the M&M section.

5. We believe that the authors should back-up the protein expression data of human insulin with quantitative RNA analyses as well.

6. Could the authors purify splenocytes from diabetic +/+, KI/+ and KI/KI mice and transfer them to NOD.Scid to further evidence the autoreactive nature of T cells?

7. Could they evaluate Treg frequency and numbers and perhaps perform Treg transfers from KI/KI mice to +/+?

8. What do the authors think will be the phenotype of KI mice crossed to ins2-deficient background? Are they planning on it? Could they discuss it?

9. Could they discuss other similar models (ref 33) further and perhaps how their model could be improved, for example by expressing human HLA?

10. It is not clear how the PCR analyses were done (lines 168-170).

**********

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PLoS One. 2019 Dec 10;14(12):e0225021. doi: 10.1371/journal.pone.0225021.r002

Author response to Decision Letter 0

20 Nov 2019

RE: PONE-D-19-29642

“Replacing murine insulin with human insulin protects NOD mice from diabetes”

Response to the reviewers’ comments

Dear Editor,

First, we’d like to thank the Academic Editor and the reviewers for their constructive and thoughtful comments on this manuscript. Below, we address each of the points raised. The person/people who made these comments is/are show in in parenthesis. The comment, or a precis of it, is shown in bold and our response in plain text. The changes to the manuscript text are highlighted in red text on the tracked change version.

1. (Academic Editor and Reviewer 1) Are there any off-target effects of the CRISPR/Cas9?

We are confident that the protection that we observe in NOD mice is not attributable to off-target genomic mutations caused by CRISPR/Cas 9 for the following reasons.

1. The mutation was back crossed to NOD mice for three generations. Each back cross reduces the load of putative off target mutations by 50%, so after three generations >87% of putative off-target mutations would have been lost.

2. There was no difference in the diabetes incidence of mice from different branches of the pedigree, suggesting that the phenotype was due to the alteration of the INS1 locus not elsewhere in the genome. We saw no difference between litters; if putative mutations were segregating and responsible for the phenotype, we would have expected to see differences between litters which we did not. The wildtype mice were a mix of littermate controls and NOD mice from our colony. There were no differences between these two groups of control mice. If there was an off-target or background effect, then we would have seen it there too.

3. Off-target effects of CRISPR have been reported to be lower in vivo than similar experiments in vitro [1]. Furthermore, the putative CRISPR/Cas9 introduced mutations should be considered in the context of the background mutation rate per mammalian generation has been estimated to be 70-175 de novo mutations per diploid genome per generation [2, 3].

4. The single guide RNAs had relatively high off-target scores on IDT’s algorithm (https://sg.idtdna.com/site/order/designtool/index/CRISPR_SEQUENCE). The sgRNAs used that led to the mouse in the study had off target scores of 47 and 67, indicating that they have a modest risk of off target effects. These scores are now included in Supplementary Table 1B.

5. In addition to the points raised above and given insulin’s well-established role in the immune pathogenesis of diabetes, both in humans and NOD mice it is extremely unlikely that protection from diabetes would develop in CRISPR-modified mice due to a mutation in an unrelated gene.

The possible role of off target mutations is now discussed on page 5, lines 97-100 and in the Discussion (page 13, lines 256-265).

2.(Reviewer 1) What do the authors mean by 'most of the amino acid coding region' being replaced (l142, p8)?

Because the final 16 amino acids in the A-chain of murine insulin 1 and human insulin are identical this part of the gene was not modified. This has now been clarified on page 9, lines 163-165.

3. (Reviewer 1) The antibodies used for ELISA and IHC to detect mouse vs human insulin: how certain are the authors that there is no cross-reactivity?

We have used well established and characterized mAbs and appropriate controls to exclude the possibility of any cross reactivity between mouse and human (pro)insulin.

1. For IHC, the mAb used for histology has been extensively characterized and shown to be specific for human insulin [4]. Furthermore, we did not detect any staining in sections from mice that did not express human insulin.

2. For ELISAs we used a commercial ELISA kit from Mercodia ELISAs which is specific for human insulin. The mAbs used in the ELISA assays for mouse insulin do cross react with human and murine insulin. However, human insulin can be measured without cross reactivity with murine insulin. To determine the concentration of murine insulin the human insulin component is subtracted, according to the manufacturer’s protocol. This is now described more clearly in Methods section.

4.(Reviewer 2) How do the authors explain the fact that KI/+ mice have the same incidence of T1D as KI/KI mice?

The simplest interpretation of this observation is that the expression of human insulin, in place of murine insulin 1 induces dominant protection against autoimmune diabetes. At this stage it is not known whether this is mediated by a Treg population, or impacts upon thymic selection, or another pathway. A thorough analysis of the mechanism(s) underlying the reduced incidence of autoimmune diabetes in this model would require several years work and is therefore beyond the scope of the current manuscript.

5. Discuss more the similarities and differences in the amino acid sequence between mouse ins1, ins2 and human insulin, with regards B:9-23 and Maki Nakayama’s paper in Nature 2004

The amino acid sequence of insulin B9-23 epitope is identical between murine insulin 1, murine insulin 2 and human insulin (see Figure 1). All have the pivotal B16 tyrosine residue that was mutated to alanine in Dr Nakayama’s 2005 Nature paper (Ref No 9 in the manuscript). Figure 1 highlights that the most difference in amino acid sequence are found in the C-peptide of proinsulin. This is consistent with reports that this region, in addition to B9-23, harbors epitopes important for the immune mediated destruction of beta cells in the NOD mouse model [5]. This is now discussed in the Discussion (p14-15, lines 300-308).

6. The authors need to measure mouse ins1 and 2 as well human insulin in the thymus of +/+, KI/+ and KI/KI mice.

As noted above a thorough analysis of the mechanism of diabetes suppression in this model is beyond the scope of the current manuscript. From our genetic analysis we know that the HuPI KI mice have human insulin in place of murine insulin 1. For this reason, KI/KI mice will not have any murine insulin 1 in the thymus. We have not modified the murine insulin 2 locus, so this will not be altered. We assume that human insulin, like murine insulin 1, will be almost exclusively detected in the pancreatic beta cells consistent with the data we have presented.

7. (Reviewer 2) Statistics are missing from the M&M section

Thank you for drawing this to our attention, we have now included a description of the statistical analysis into the relevant parts of the Methods section.

8. (Reviewer 2) The authors should back-up the protein expression data of human insulin with quantitative RNA analyses

With respect, we disagree. We have already shown that mRNA for human insulin is expressed in the islets of the HuPI mice (Figure 2C). We have also shown that both human insulin protein and C-peptide are expressed. In our view, the critical point is that human insulin and C-peptide are expressed, albeit at variable concentrations. In our view measuring the mRNA levels will not give further insights above what have already be reported.

9. (Reviewer 2) Could the authors:

Purify splenocytes from diabetic +/+, KI/+ and KI/KI mice and transfer them to NOD.Scid?

Evaluate Treg frequency and numbers and perhaps perform Treg transfers from KI/KI mice to +/+?

We thank Reviewer 2 for these helpful suggestions. We are planning a manuscript which will address the mechanism(s) by which HuPI mice are protected from diabetes. However, to come to a clear conclusion regarding the mechanism is beyond the scope of this manuscript. Here our aim is to report the generation and evaluation of HuPI mice.

10. Could they discuss other similar models (ref 33) further and perhaps how their model could be improved, for example by expressing human HLA?

A more detailed discussion of other models has now been included in the Discussion (p15-16 line 321-338). We also discuss the further development of this model, including the expression of HLA and human (pro)insulin specific TCRs.

11. It is not clear how the PCR analyses were done (lines 168-170).

Thank you for drawing this to our attention. In addition to the primers’ sequences we have now included more details about how the PCR was performed in the Materials and Methods section of the manuscript (p6, line 113-120).

References cited

1. Yoshimi K, Kaneko T, Voigt B, Mashimo T. Allele-specific genome editing and correction of disease-associated phenotypes in rats using the CRISPR-Cas platform. Nature communications. 2014;5:4240. Epub 2014/06/27. doi: 10.1038/ncomms5240. PubMed PMID: 24967838; PubMed Central PMCID: PMCPMC4083438.

2. Nachman MW, Crowell SL. Estimate of the mutation rate per nucleotide in humans. Genetics. 2000;156(1):297-304. Epub 2000/09/09. PubMed PMID: 10978293; PubMed Central PMCID: PMCPMC1461236.

3. Roach JC, Glusman G, Smit AF, Huff CD, Hubley R, Shannon PT, et al. Analysis of genetic inheritance in a family quartet by whole-genome sequencing. Science. 2010;328(5978):636-9. Epub 2010/03/12. doi: 10.1126/science.1186802. PubMed PMID: 20220176; PubMed Central PMCID: PMCPMC3037280.

4. Asadi A, Bruin JE, Kieffer TJ. Characterization of Antibodies to Products of Proinsulin Processing Using Immunofluorescence Staining of Pancreas in Multiple Species. J Histochem Cytochem. 2015;63(8):646-62. Epub 2015/07/29. doi: 10.1369/0022155415576541. PubMed PMID: 26216140; PubMed Central PMCID: PMCPMC4530395.

5. Levisetti MG, Lewis DM, Suri A, Unanue ER. Weak Proinsulin Peptide–Major Histocompatibility Complexes Are Targeted in Autoimmune Diabetes in Mice. Diabetes. 2008;57(7):1852-60. doi: 10.2337/db08-0068.

Attachment

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Decision Letter 1

Matthias G von Herrath

22 Nov 2019

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

PONE-D-19-29642R1

Dear Dr. Mannering,

We are pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it complies with all outstanding technical requirements.

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Matthias G von Herrath, MD PhD

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Good paper! :-)

Reviewers' comments:

Acceptance letter

Matthias G von Herrath

2 Dec 2019

PONE-D-19-29642R1

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

Dear Dr. Mannering:

I am pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department.

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on behalf of

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Associated Data

    This section collects any data citations, data availability statements, or supplementary materials included in this article.

    Supplementary Materials

    S1 Fig. Sequence of the homology-directed repair (HDR) template.

    (PDF)

    Click here for additional data file. (39KB, pdf)
    S2 Fig. Human insulin is localized to the pancreatic islets in NOD.HuPI mice.

    (PDF)

    Click here for additional data file. (6.4MB, pdf)
    S3 Fig. NOD.HuPI mice exhibit delayed insulitis (100d).

    (PDF)

    Click here for additional data file. (89.5MB, pdf)
    S4 Fig. NOD.HuPI mice exhibit delayed insulitis (300d).

    (PDF)

    Click here for additional data file. (92.8MB, pdf)
    S1 Table. Production of human insulin knock-in mice by CRISPR/Cas9 mutagenesis.

    (PDF)

    Click here for additional data file. (70.5KB, pdf)
    S2 Table. Molecular confirmation of INS knock-in.

    (PDF)

    Click here for additional data file. (59KB, pdf)
    S3 Table. Genotype ratios in offspring of NOD.HuPI mice.

    (PDF)

    Click here for additional data file. (44.6KB, pdf)
    S4 Table. Insulin-specific primers.

    (PDF)

    Click here for additional data file. (70.8KB, pdf)
    S1 Raw Image. Original gel image of Fig 2C.

    (TIF)

    Click here for additional data file. (6.1MB, tif)
    Attachment

    Submitted filename: R2R.docx

    Click here for additional data file. (39.6KB, docx)

    Data Availability Statement

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