WO2025221974A1

WO2025221974A1 - Hsa-mir-amc1 inhibitor/antagomir and method of treatment

Info

Publication number: WO2025221974A1
Application number: PCT/US2025/025125
Authority: WO
Inventors: C. Michael Dipersio; Allen GERBER; Salima Sadruddin LALANI
Original assignee: Albany Medical College
Current assignee: Albany Medical College
Priority date: 2024-04-19
Filing date: 2025-04-17
Publication date: 2025-10-23
Anticipated expiration: 2026-10-19

Abstract

A newly discovered microRNA, hsa-miR-AMC1, serves as an early biomarker of coxsackievirus B4-induced type 1 diabetes. In addition, inhibiting hsa-miR-AMC1 may provide therapeutic benefits to type 1 diabetes patients. Trophoblast cells were also found to be a reliable model for identifying microRNAs that might be useful diagnostic markers or therapeutic targets for coxsackievirus B-induced type 1 diabetes

Description

TITLE hsa-miR-AMCl INHIBITOR/ ANTAGOMIR AND METHOD OF TREATMENT

BACKGROUND OF THE INVENTION

1. FIELD

[0001] The present disclosure relates to coxsackievirus B-induced type 1 diabetes and more particularly, to a microRNA inhibitor and method of viral inhibition using the inhibitor.

2. DESCRIPTION OF THE RELATED ART

[0002] Type 1 diabetes (T1D) is an autoimmune disease caused by persistent immune-mediated destruction of insulin-producing pancreatic [:1 cells. It is estimated that 1/300 people in the United States develop T1D by the age of 18 years (JDRF). Currently, there is no prevention or cure for T1D, which can only be managed with life-long insulin supplements, immunotherapies, or islet cell transplants. T1D is a complex disease, wherein a combination of genetic and environmental factors interact to trigger immune-mediated destruction of pancreatic [:1 cells. Numerous clinical observations suggest a causal link between coxsackievirus B (CVB) infection and the onset of T1D. For example, studies have shown that if a pregnant woman is infected with CVB, her child will have increased risk of developing T1D. Moreover, case studies have shown that the onset of diabetes often occurred during an active CVB infection. Other studies have shown that patients with newly diagnosed T1D often have higher levels of CVB antibodies, or that CVB infection persists in T1D patients. Individuals whose siblings have T1D are more likely to develop T1D following a CVB infection, indicating an underlying genetic component.

[0003] The epidemiological association of CVB with T1D suggests that treating CVB infection to reduce viral load could delay or prevent the onset of T1D. Indeed, mounting evidence from preclinical and clinical studies supports a causal role of viral infection in triggering T ID. For example, one study showed that non-obese mice were protected from coxsackievirus B4 (CVB4)-induced T1D by treatment with a monovalent vaccine against conserved regions of viral protein 1 in CVB4. The Pro vention Bio PRV-101 multivalent vaccine study to target CVB is currently in clinical trials (NCT04690426) for prevention of T1D (Provention Bio). More recently, Krogvold and coworkers showed in a randomized phase II clinical trial that insulin levels in newly diagnosed T1D patients were preserved following treatment with antiviral agents. These findings indicate that antiviral strategies have potential to prevent or treat T1D. It follows that endogenous genetic elements that support or promote CVB infection, such as transposable elements and microRNAs (miRNAs), may represent novel targets to inhibit virus-induced T1D. [0004] miRNAs are non-coding RNAs that can regulate gene expression through inhibition of mRNA translation. The relationship of miRNAs to viral immunity implicates them in CVB mediated autoimmunity associated with T1D. Moreover, some miRNAs are implicated in the etiology of T1D. For example, miRNA-146a is differentially expressed in T1D patients and contributes to diabetic complications by regulating the inflammatory response. Similarly, miR-184-3p is enriched in insulin-producing pancreatic [:1 cells where it regulates several [:1 cell functions. Importantly, many miRNAs are differentially regulated in the placenta during viral infection and have been implicated in providing antiviral immunity to the fetus. Thus, a better understanding of the early immune response may reveal important clues regarding how response to viral infection later in life (e.g., in pancreatic [:1 cells) may lead to T1D. In particular, trophoblast cells that comprise the epithelial cell compartment of the placenta may provide a useful model for investigating how the prenatal immune system is altered during viral infection, and how certain miRNAs are impacted by and/or alter viral infection.

[0005] The epidemiological association of coxsackievirus B infection with type 1 diabetes suggests that therapeutic strategies that reduce viral load could delay or prevent disease onset. Moreover, recent studies suggest that treatment with antiviral agents against coxsackievirus B may help preserve insulin levels in type 1 diabetic patients. Accordingly, there is a need in the art for early biomarkers of coxsackievirus B4-induced type 1 diabetes and inhibition targets to provide therapeutic benefit to type 1 diabetes patients.

BRIEF SUMMARY OF THE INVENTION

[0006] The present invention involves the use of a newly discovered miRNA, hsa- miR-AMCl, as a biomarker for coxsackievirus B4-induced type 1 diabetes and an inhibition target to provide therapeutic benefit to type 1 diabetes patients. Small RNA-sequencing was used to show that infection of immortalized trophoblast cells with coxsackievirus caused differential regulation of several miRNAs. One of these, hsa-miR-AMC 1 , was similarly upregulated in human pancreatic [:1 cells infected with coxsackievirus B4. Moreover, treatment of [:1 cells with non-cytotoxic concentrations of an antagomir that targets hsa-miR- AMC 1 led to decreased CVB4 infection, suggesting a positive feedback loop wherein this microRNA further promotes viral infection. Consistently, treatment of coxsackievirus B4- infected [:1 cells with the hsa-miR-AMC 1 antagomir was associated with a trend toward increased insulin secretion. hsa-miR-AMC 1 may thus serve as a potential early biomarker of coxsackievirus B4-induced type 1 diabetes and inhibiting hsa-miR-AMC 1 may provide therapeutic benefit to type 1 diabetes patients. The use of trophoblast cells was also shown to be a model for identifying microRNAs that might be useful diagnostic markers or therapeutic targets for coxsackievirus B-induced type 1 diabetes.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

[0007] The present invention will be more fully understood and appreciated by reading the following Detailed Description in conjunction with the accompanying drawings, in which:

[0008] FIG. 1 is a graph showing that hsa-miR-AMCl is upregulated in pancreatic P cells following infection with the CVB4 serotype, but not with any of the other five CVB serotypes. Cells were left uninfected or were infected with the CVB 1-6 serotypes, including a prototype strain of CVB4 (CVB4-JVB) and a diabetogenic strain of CVB4 (CVB4-E2), as indicated. qPCR was performed to determine relative hsa-miR-AMC 1 expression after normalization to RNU48 expression (a small-nucleolar RNA control). hsa-miR-AMCl expression in uninfected cells is set at a value of 1.0. Data are average ± S.E.M; n = 3; *p < 0.05, **p < 0.01 , t-test.

[0009] FIG. 2 is a pair of graphs showing inhibition of pancreatic P cell infection by

(A) CVB4-JVB or (B) CVB4-E2 following treatment with non-cytotoxic concentrations of hsa-miR-AMCl antagomir, determined by plaque assays using LLCMK2 cells. For (A) and

(B), infectivity in the absence of antagomir is set at 100%. Data are average ± S.E.M.; n = 3 biological replicates; ****p < 0.0001, One-way ANOVA followed by post hoc Tukey's multiple comparisons test.

[0010] FIG. 3 is a series of graphs showing the effects of hsa-miR-AMCl antagomir on glucose-stimulated insulin secretion (GSIS) in (A) non-infected, (B) CVB4-JVB-infected, and (C) CVB4-E2-infected pancreatic P cells. Data are average ± S.E.M.; n = 3 biological replicates; *p < 0.05, ** p < 0.001, *** /? < 0.0001, One-way ANOVA followed by post hoc Tukey's multiple comparisons.

[0011] FIG. 4 is a graph showing that the antagomir against hsa-miR-AMCl is not cytotoxic in pancreatic P cells. Cell viability was determined using an MTS assay to measure absorbance at 490 nm with a microplate reader. The dotted line represents the 90% viability cutoff criteria. Data are presented as average viability ± S.E.M from three biological replicates.

[0012] FIG. 5 shows (A) a Venn diagram to indicate overlap of 13 genes between predicted hsa-miR-AMC 1 target genes and differentially expressed genes (DEGs) in C VB4- E2-infected pancreatic P cells, and (B) a heatmap of differential expression of these 13 overlapping genes in CVB4-E2 infected pancreatic [:1 cells compared with uninfected (control) cells (n=3).

[0013] FIG. 6 is a graph showing that infection with the CVB4-E2 strain leads to the down-regulation of the gene encoding Leucine-rich repeat LGI family member 3 (LGI3 , which is a predicted gene target of hsa-miR-AMCl. Furthermore, it shows that insulin-like growth factor binding protein 5 (IGFBP5), which is a gene known to be induced by LGI3, is similarly down-regulated during CVB4-E2 infection. qPCR was performed to determine relative gene expression after normalization to GAPDH expression (a housekeeping control gene). GAPDH expression in uninfected cells is set at a value of 1.0. Data are average ± S.E.M; n = 3; *p < 0.05, ***p < 0.001 t-test. Primer sequences are as follows: LGI3: Forward GATTGGAGACAACGCCTTCA (SEQ ID NO: 1), Reverse CTCGGAAGGTGAACTTGGATAG (SEQ ID NO: 2). IGFBP5'. Forward TGAGATGAGACAGGAGTCTGAG (SEQ ID NO: 3), Reverse GTCACAATTGGGCAGGTACA (SEQ ID NO: 4). GAPDH: Forward CGGAGTCAACGGATTTGGTCGTAT (SEQ ID NO: 5), Reverse GCAGGTCAGGTCCACCACTGA (SEQ ID NO: 6).

DETAILED DESCRIPTION OF THE INVENTION

[0014] Referring to the figures, wherein like numerals refer to like parts throughout, the present invention was premised on the identification of a novel miRNA, hsa-miR-AMCl, that enhances CVB4 infection of pancreatic [:1 cells and may also reduce insulin production by pancreatic [:1 cells infected with certain diabetogenic CVB4 strains. Based on this identification, it was hypothesized that CVBs alter the miRNA signature of pancreatic [:1 cells, which may in turn lead to alterations in the transcriptome that contribute to the autoimmune response that triggers T1D. Such a role would implicate these miRNAs as therapeutic targets for inhibiting CVB infection or its downstream effects to delay or prevent T1D. More specifically, the infection of trophoblast cells with CVB4 leads to the dysregulation of several miRNAs. One of these miRNAs, hsa-miR-AMCl, is up-regulated in human pancreatic [:1 cells (a primary target cell of CVBs) following CVB4 infection.

[0015] The present invention comprises the treatment of [:1 cells with an antagomir to inhibit hsa-miR-AMC 1 , which was shown to lead to decreased CVB4 infection and increased insulin production. The sequence of the antagomir is as follows: 5’-mG / ZEN / mGmC mCmUmC mAmCmA mCmCmG mUmCmC mAmCmA /3ZEN/-3’ (SEQ ID NO: 7). hsa- miR-AMC 1 has therefore been identified as a novel miRNA that is up-regulated by CVB4 infection of pancreatic [:1 cells where it further promotes infection and suppresses insulin secretion. This miRNA may thus serve as a novel diagnostic marker and/or therapeutic target for CVB4-induced T1D.

[0016] In connection with the present invention, two known and three novel miRNAs that are differentially expressed during CVB4 infection of trophoblast cells were considered. hsa-miR-1304-5p was down-regulated during infection, while the other four miRNAs were significantly up-regulated. Interestingly, five target genes of miRNA hsa-miR-3913 (TLR8, SIRPG, TLR7, FUT2, and SMARCE1) have been identified as risk factors of T1D, as documented in Genome-wide association studies (GWAS). Moreover, all five of the latter genes were directly or indirectly associated with an immune response to viral infection. Interestingly, hsa-miR-AMC3 has a predicted target gene, CTLA4, wherein a single nucleotide polymorphism shows a strong association with T1D risk due to autoimmune susceptibility.

[0017] From among the several miRNAs that were determined to be altered by CVB infection, hsa-miR-AMCl was investigated because (1) this novel miRNA has not yet been explored in any disease context, and (2) the pathway enrichment analysis for predicted hsa- miR-AMCl target genes revealed overlap with target genes of hsa-miR-184, a known negative regulator of genes that control insulin in pancreatic [:1 cells. Furthermore, enriched transcription factor and protein-protein interactions analysis of hsa-miR-AMCl identified association with nuclear factor, erythroid 2 (NFE2), a known regulator of the heme oxygenase 1 (HM0X1) gene for which down-regulation is associated with delayed onset of T1D, as well as with nuclear respiratory factor 1 (Nrfl), for which decreased function is associated with loss of P cell activities.

[0018] Thirteen predicted target genes of hsa-miR-AMCl that were also differentially expressed by pancreatic [:1 cells infected with the diabetogenic CVB4-E2 strain were identified (Fig. 5a, Table 3). Of these, TNS3, MAFK, GALNT10, UBE3B, and Clorf21 are also predicted target genes of hsa-miR-184. This subset of thirteen genes enriched the insulin/IGF-MAPK cascade in Panther pathways. Furthermore, most of these genes were down-regulated with the exception of SLC7A5, MAFK, TNS3, and ZNF707 (Fig. 5b). Notably, an increase in transcription factor MAFK has been identified to hamper pancreatic beta cell function.

[0019] To understand the potential link between CVB infection and insulin regulation, we assessed overlap/interaction between pathways regulated by CVB and insulin regulatory pathways. Indeed, CVB viruses hijack several host pathways such as phosphoinositide-3-kinase-protein kinase B/Akt (PI3K/Akt) pathway, and mitogen-activated protein kinase (MAPK) to gain a replicative advantage. The MAPK pathway controls carbohydrate metabolism and cell growth via insulin receptors and insulin-like growth factor receptors, respectively. The MAPK pathway could be initiated via enteroviral modulation of the PI3K/Akt pathway, which is used by enteroviruses in the early stages of infection to promote viral replication and decrease apoptosis. On the other hand, AMP-activated protein kinase (AMPK) is a well-identified target for diabetes and diabetes-related symptoms and a kinase regulator of energy homeostasis. AMPK is necessary for activation of Akt and is modulated by viruses during an infection. One hypothesis is that under mitochondrial stress caused by enterovirus infection there is increased viral replication and inhibition of IFN immune response via downstream AMPK targets. Considering that hsa-miR-AMCl may regulate the insulin/IGF pathway-MAPK cascade in pancreatic [:1 cells infected with CBV, it is interesting to speculate that its antagomir could prevent collapse of this cascade. Consistently, it was observed in cells infected with the clinical diabetogenic CVB4-E2 variant that treatment with the hsa-miR-AMC 1 antagomir increased glucose-dependent induction of insulin secretion above the level of induction in untreated cells (Fig. 3).

[0020] Finally, the use of the trophoblast cell model is an exciting development that may have important utility in the research of CVB-induced-TID. Indeed, there has been recent interest in investigating how maternal T1D impacts placental function and fetal development. The finding that the trophoblast miRNA, hsa-miR-AMC 1 , is also present in pancreatic [:1 cells and was significantly upregulated during CVB4 infection leads us to propose that the trophoblast cell model may be useful for discovering novel biomarkers with postnatal relevance to T ID. In summary, the present invention identified a novel miRNA, hsa-miR-AMC 1 , that enhances CVB4 infection of pancreatic [:1 cells and may also reduce insulin production by pancreatic [:1 cells infected with certain diabetogenic CVB4 strains. EXAMPLE

[0021] Cell lines

[0022] The immortalized pancreatic [:1 cell line, EndoC-PH 1 , was purchased from Human Cell Design (HCD) and grown in PCOAT (HCD, Cat. # BC-120) or Matrigel- fibronectin (100 pg/mL Coming, Cat. #CLS356234 and 2pg/mL, Sigma, Cat. #F1141, respectively), in OPTI01 medium (HCD, Cat. # OBI-100). Cells were passaged every 7 days. The immortalized human trophoblast cell line hTERT (Sw.71), was purchased from Abmgood (Cat. #T0532) and grown on dishes coated with Applied Cell Extracellular Matrix (Abmgood, Cat. #G422) in PriGrow IV medium (Abmgood, Cat. #TM004) supplemented with 10% fetal bovine serum (FBS, GeminiBio, Cat. #100-106), 1% L-glutamine (Corning, Cat. #25-005-CI), 10 mM HEPES (Sigma- Aldrich, Cat. #83264), 0.1 mM MEM non- essential amino acids (Sigma- Aldrich, Cat#M7145), 1 mM sodium pyruvate (GIBCO, Cat. #11360-070) and 1% penicillin-streptomycin (PSA, GIBCO, Cat. #15140-100 122). LLC- MK2 derivative cells (ATCC# CCL-7.1) or Vero cells (ATCC# CCL-81) were grown in 101 Eagle’s Modified Essential Medium (EMEM, ATCC, Cat. #30-2003) supplemented with 10% FBS (GeminiBio, Cat. #100-106) and 1% PSA (GIBCO, Cat. #15140-122). All cells were grown at 37°C, 5% CO2.

[0023] Propagation of viruses

[0024] Coxsackievirus (CVB) serotypes 1, 2, 3 4, 5 and 6 (ATCC# VR-28, ATCC# VR-29, ATCC# VR-30, ATCC# VR-184, ATCC# VR-185, ATCC# VR-155, respectively) were propagated in LLC-MK2 derivative cells (ATCC# CCL-7.1) or Vero cells (ATCC# CCL-8.1). CVB4-E2 (diabetogenic strain) was obtained from Laboratoire de Virologie ULR3610, ULille, and CHU Lille, France. Briefly, a 70% confluent monolayer of LLC-MK2 or Vero cells was infected for 1 hour with CVBs at a multiplicity of infection (MOI) of 0.1 or 0.01. The inoculum was then removed, and cells were washed with phosphate-buffered saline (Coming, Cat. #21-040-CV) and maintained in EMEM supplemented with 2% FBS and 1% PSA. Cytopathic effects (CPE) were monitored over 1-5 days or until 90% CPE was observed. The viruses were harvested by 3 -freeze-thaw cycles and contents were collected and centrifuged at 10,000xg at 4°C for 10 minutes. The supernatant was collected, aliquoted, and stored at -80°C. Viral titration was performed using plaque assays.

[0025] Antagomirs

[0026] A customized antagomir against hsa-miR-AMCl miRNA (Integrated DNA

Technologies) was dissolved in DNase/RNase-free water (100 pM or 500 pM) and aliquoted for storage at -80°C.

[0027] RNA sequencing and bioinformatics

[0028] Small RNA sequencing (RNA-Seq)

[0029] Sw.71 cells were seeded (3xl0⁵ cells/well) overnight and then infected with

CVB4-JVB strain for 24 hours at a multiplicity of infection (MOI) of 1. Infected cells were washed and pelleted for RNA extraction, library preparation, and small RNA-Seq. Reads were aligned to miRbase (miRNA), and differential gene expression was performed using DESeq2. For novel miRNA prediction, sequences were aligned to the human genome and subjected to RNA folding and secondary structure analysis (miRDeep2, V2_0_0_7). All experiments were performed in three biological replicates (i.e., in triplicate).

[0030] Standard RNA-Seq [0031] Sw.71 cells (3xl0⁵/well) or EndoC-PHl cells (6.72xl0⁵/well) were seeded overnight and then infected with CVB4 (CVB4-JVB or -E2 strain) for 24 hours at a MOI=1 (Sw.71 cells) or for 1 hour at a MOI=0.1 (EndoC-130 0H1 cells). Infected cells were washed and pelleted for RNA extraction (RNeasy Plus Kit, Cat. #74136), library preparation, and RNA-Seq. Reads were aligned to the human reference genome, Genome Reference Consortium Human Build 38, with STAR 2.7.10b. Differential gene expression was performed using DESeq2 1.40.2. All experiments were performed in triplicate.

[0032] miRNA isolation and qPCR

[0033] miRNA was extracted from infected cells using a mirVana™ miRNA isolation kit (ThermoFisher Scientific, Cat. #AM1560). Reverse transcription was performed using TaqMan™ MicroRNA Reverse Transcription Kit (ThermoFisher Scientific, Cat. #4366596) and TaqMan™ MicroRNA Assay (ThermoFisher Scientific, Cat. #4427975, assay IDs 001006 and CT9HJTV) using a custom-designed looped RT primer to produce cDNA specific to small-nucleolar RNA “RNU48” (control miRNA) or hsa-miR-AMC 1 , according to the manufacturer’s instruction. qPCR was performed using TaqMan™ Fast Advanced Master Mix (ThermoFisher Scientific, Cat. #4444557) in a BioRad CFX96 TouchTM Real- Time PCR Detection System. Fold-change in miRNA expression was calculated using the 2- AACT method. All experiments were performed in triplicate.

[0034] RNA isolation and qPCR

[0035] For target gene analysis, EndoC-0Hl cells (6.72xl0⁵/well) were seeded overnight then infected with CVB4 (CVB4-JVB or -E2 strain) for 1 hour at a MOI=0.1 (EndoC-PHl cells). Infected cells were washed and pelleted for RNA extraction (RNeasy Plus Kit, Cat. #74136). Reverse transcription was performed using iScript™ cDNA Synthesis Kit (Bio-Rad, Cat. #1708890) according to the manufacturer’s instruction. qPCR was performed using Sso Advanced Universal SYBR Green Supermix (Bio-Rad, Cat. 1725271) in a BioRad CFX96 TouchTM Real-Time PCR Detection System using the manufacturer’s protocol. Fold-change in gene expression was calculated using the 2-AACT method. All experiments were performed in triplicate.

[0036] Cytotoxicity analysis

[0037] To evaluate cytotoxicity of the hsa-miR-AMC 1 antagomirs on EndoC-PHl cells, the CellTiter 96 Aqueous One Solution Cell Proliferation MTS assay kit (Promega, Cat. #G3582) was used according to the manufacturer’s instructions. Briefly, 2.24xl0⁴ cells/well were seeded in 96-well plates pre-coated with Matrigel-150 fibronectin matrix, as described above. The antagomir was serially diluted (10 pM - 0.1 pM) and transfected into EndoC-PHl cells using OptiMEM media (Gibco, Cat. #31985062) and Lipofectamine RNAiMAX Transfection Reagent (Invitrogen, Cat. #13778150) following the manufacturer’s instructions. After 3 hours transfection mix was removed and replaced with EndoC-PHl growth medium. Cells were incubated at 37°C for 24h, then MTS solution was added to each well and incubated for an additional 2 hours for color development. Plates were read at 490 nm and cytotoxicity was plotted using GraphPad Prism (v.9.5.1). All experiments were performed in triplicate.

[0038] Viral inhibition analysis

[0039] To evaluate effects of miRNA inhibition, EndoC-PH 1 cells grown on 96-well plates were transfected with the hsa-miR-AMC 1 antagomir 24 hours before infection with CVB4-JVB or CVB4-E2 strains (MOI=0.1). After incubation for 1 hour, the inoculum was removed, and cells were incubated in EndoC-PHl cells growth medium for another 24 hours. Supernatants were collected, and plaque assays performed to determine viral infectivity. The percentage of viral inhibition was plotted using GraphPad Prism (v.9.5.1). All experiments were performed in triplicate.

[0040] Glucose-stimulated insulin secretion (GSIS) analysis

[0041] GSIS was performed as described by Tang et al. with minor modifications. Briefly, EndoC-PHl cells were transfected with the antagomir or mock-transfected without the antagomir for 24 hours, then infected with CVB4-E2 or CVB4-JVB strain for 1 hour or left uninfected as a control. The next day cells were serum-starved, first in serum-free medium for 1 hour followed by Krebs buffer solution for 1 hour (Human Cell Design, PKREBS®). Cells were then stimulated with 20 pM glucose for 40 minutes and supernatant was collected. Samples were stored at -20°C prior to enzyme-linked immunosorbent assay (ELISA) to quantify insulin secretion (Human Insulin Kit, Mercodia, Cat. #10-1113-01). ELISA was performed on duplicate samples from each of three independent experiments.

[0042] Statistical analysis

[0043] Data are presented as average ± S.E.M from three biological replicates (n=3), with at least two technical replicates for each experiment. Statistical significance was determined using a t-test or one-way ANOVA, as detailed in figure legends; *p < 0.05, **p < 0.005, ***p < 0.001, ****p < 0.0001.

[0044] Results

[0045] Identification of differentially expressed miRNAs in CVB4-infected trophoblast cells. [0046] To identify miRNAs that might regulate T ID-associated genes following CVB infection, Sw.71 cells were infected with a prototype/reference strain of CVB4, CVB4-JVB, for 24 hours then isolated total RNA and performed miRNA sequencing. Table 1 shows the top 5 differentially expressed miRNAs in response to CVB4-JVB infection, compared to uninfected cells.

Table 1 - Top 5 differentially expressed miRNAs in CVB4-infected trophoblast (Sw.71) cells (n=3).

*Log CPM = logarithm of counts per million reads. ⁺Novel miRNAs

[0047] The novel miRNAs were predicted using hairpin structures of the precursor miRNAs. The sequences of the novel miRNAs are as follows: [0048] The miRanda (v3.3a) target scanner was used to predict target sites based on miRNA sequences and corresponding genomic cDNA sequences. As an example, predicted target genes of hsa-miR-AMCl are listed in Table 2.

Table 2 - Predicted hsa-miR-AMCl target genes

ATF13A1 IGSF9B SLC7A5 I F2R

[0049] hsa-miR-AMCl is expressed in pancreatic 0 cells.

[0050] hsa-miR-AMC 1 was further investigated since this novel miRNA has not been explored in any disease context. Interestingly, pathway enrichment analysis of predicted hsa- miR-AMC 1 target genes (Table 2) revealed a 5.6-fold enrichment of predicted hsa-miR-184 target genes (false discovery rate, 4.7E-02). This finding is intriguing, as hsa-miR-184 is expressed in pancreatic 0 cells where it negatively regulates genes involved in insulin production, some of which are involved in T1D. To determine whether hsa-miR-AMCl is also expressed in pancreatic 0 cells, and whether its expression is altered by infection with different CVB serotypes, cells were infected with each of six CVB serotypes (CVB1-6), including both a prototype variant of CVB4 (CVB4-JVB) and a clinical diabetogenic variant of CVB4 (CVB4-E2), and qRT-PCR of uninfected cells was performed. The results showed that not only is hsa-miR-AMC 1 expressed in pancreatic 0 cells, but it is significantly up- regulated during infection with either the prototype or diabetogenic variant of CVB4, but not with the other 5 CVB serotypes (Fig. 1), indicating a serotype-specific response. [0051] Inhibition of hsa-miR-AMCl reduces CVB4 infection of pancreatic 0 cells.

[0052] Pancreatic 0 cells are a known site of postnatal CVB4 infection and replication. Since it was observed that hsa-miR-AMCl was highly up-regulated in CVB4- infected cells (Table 1; Fig. 1), the effect of a hsa-miR-AMCl inhibitor (antagomir) on CVB4 infectivity of 0 cells was evaluated. A cell viability assay showed that treatment with the hsa- miR-AMCl antagomir was well tolerated by pancreatic 0 cells over a range of concentrations (0.1 pM to 10 pM) (Fig. 4). Interestingly, treatment with the hsa-miR-AMCl antagomir significantly inhibited infection of pancreatic 0 cells (p <0.0001) by either the prototype CVB4-JVB strain (Fig. 2a) or the diabetogenic CVB4-E2 variant (Fig. 2b). Taken together, the findings indicate that hsa-miR-AMC 1 is induced by CVB4 infection in pancreatic 0 cells and promotes CVB4 infection. The initial induction of hsa-miR-AMCl upon CVB4 infection may lead to altered regulation of target genes that drive a positive feedback loop to enhance infectivity.

[0053] Inhibition of hsa-miR-AMCl impacts insulin production in pancreatic 0 cells infected with the diabetogenic CVB4-E2 strain.

[0054] It was next determined if treating pancreatic 0 cells with the hsa-miR-AMC 1 antagomir alters insulin secretion in a high-glucose environment, without and with CVB4 infection. Insulin production was first determined in the absence or presence of antagomir without infection. One-way ANOVA showed that insulin secretion was increased when cells were challenged with 20 pM glucose, with or without antagomir (p = 0.0484, F = 3.133). However, Tukey’s HSD test for multiple comparisons revealed no significant difference in this induction between these two groups (p > 0.05, Fig. 3a), suggesting that antagomir treatment had no appreciable effect. Cells were next treated with antagomir followed by infection with CVB4-JVB or CVB4-E2. In CVB4-JVB infected cells without antagomir, the expected trend of increased insulin upon glucose challenge (p = 0.0001) was observed. This increase remained significant in antagomir-treated cells (p = 0.0018), although it appeared dampened (Fig. 3b). In contrast, when cells were infected with the diabetogenic CVB4-E2 variant under high glucose conditions, insulin levels were increased by antagomir treatment when compared with untreated cells (p = 0.049, Fig. 3c). This effect of antagomir-treatment on insulin levels could be due to either protection of pancreatic 0 cells from virus-induced cell death or increased ability of cells to secret insulin. In either case, these results suggest that hsa-miR-AMCl inhibition in diabetogenic CVB4 strains (e.g., E2 strain) might lead to altered gene expression that controls glucose-stimulated insulin secretion. [0055] Identification of potential hsa-miR-AMCl gene targets

[0056] The miRanda algorithm (v3.3a) was used to identify 64 genes with predicted target sites for hsa-miR-AMCl miRNA . Intriguingly, this hsa-miR-AMCl target gene set (Table 3) indicated enrichment of the “Insulin/IGF-MAPK cascade”, which regulates carbohydrate metabolism and insulin-like growth factor receptors.

Table 3 - Predicted hsa-miR-AMCl target genes that are differentially expressed in CVB4- E2 infected pancreatic [:1 cells versus uninfected cells.

Symbol log2FoldChange p-adj (False Discovery Rate)

SLC7A5 0.908602 5.48E-13

HADH -0.42553 3.21E-10

LGI3 -1.50117 1.71E-09

IGF2R -0.34264 6.22E-07

UBE3B -0.50107 1.95E-06

MAP1B -0.30186 1.43E-05

MAFK 1.064973 1.45E-05

KIDINS220 -0.25999 0.001585

GALNT10 -0.42262 0.002087

TNS3 0.333692 0.002901

MPRIP -0.27203 0.014473

ZNF707 0.55343 0.022207

Clorf21 -0.33416 0.023079

[0057] It was then asked whether any of these genes are also differentially regulated in pancreatic [:1 cells upon CVB4-E2 infection. RNA-seq followed by assessment of differential gene expression using DESeq2 revealed 2751 differentially expressed genes (DEGs) following CVB4-E2 infection (adjusted p-value < 0.05). We next determined whether any of these DEGs are predicted hsa-miR-AMCl target genes. Interestingly, thirteen genes were common between the groups (Table 3, Fig. 5a). A heat map shows the expression profile of the thirteen genes that were predicted target genes of hsa-miR-AMCl and also differentially expressed in pancreatic [I cells infected with the diabetogenic CVB4-E2 strain (Fig. 5b). Of note, some of these genes have roles in regulating glucose metabolism and insulin regulation.

[0058] Intriguingly, one of the thirteen genes, the Leucine-rich repeat LGI family member 3 (LGI3 gene, is a prognostic marker of pancreatic cancer and regulates several relevant genes. For example, LGI3 regulates insulin-like growth factor binding protein 5 (/GFBP5), which is downregulated in T1D and is associated with CVB infection in patient samples. We performed qPCR to determine the expression levels of LGI3 and its target gene IGFBP5. We showed that the infection with the CVB4-E2 strain leads to the significant down-regulation of both LGI3 and IGFBP5. Given its strong association with both T1D and CVB4 infection, LGI3 may be an interesting hsa-miR-AMCl target gene for future studies.

Claims

CLAIMS What is claimed is:

1. A compound comprising an antagomir that inhibits an miRNA selected from the group consisting of hsa-miR-AMC 1 , hsa-miR-AMC2, hsa-miR-AMC3, hsa-miR-1304- 5p, and hsa-miR-3913-5p.

2. The compound of claim 1 , wherein the miRNA is hsa-miR-AMC 1.

3. The compound of claim 2, wherein the antagomir comprises SEQ ID NO: 7.

4. A method of treating a patient, comprising the step of administering a therapeutic amount of an antagomir that inhibits an miRNA selected from the group consisting of hsa-miR-AMC 1, hsa-miR-AMC2, hsa-miR-AMC3, hsa-miR-1304-5p, and hsa- miR-3913-5p.

5. The method of claim 4, wherein the miRNA is hsa-miR-AMC 1.

6. The method of claim 5, wherein the antagomir comprises SEQ ID NO: 7.

7. The method of claim 4, wherein the patient has type 1 diabetes.

8. The method of claim 4, wherein the patient has a coxsackievirus B4 infection.

9. The method of claim 4, wherein the step of administering a therapeutic amount of the antagomir that inhibits the miRNA is performed to reduce the risk of the patient developing type 1 diabetes.

10. A method of screening a patient at risk for type 1 diabetes, comprising the steps of: detecting the presence of an miRNA selected from the group consisting of hsa-miR- AMC1, hsa-miR-AMC2, hsa-miR-AMC3, hsa-miR-1304-5p, and hsa-miR-3913-5p in a biospecimen of the patient; identifying whether the patient is a candidate for receiving an antagomir that inhibits an miRNA selected from the group consisting of hsa-miR-AMC 1, hsa-miR-AMC2, hsa-miR- AMC3, hsa-miR-1304-5p, and hsa-miR-3913-5p to lower the risk of diabetes onset; and administering a therapeutic amount of the antagomir that inhibits the miRNA selected from the group consisting of hsa-miR-AMC 1, hsa-miR-AMC2, hsa-miR-AMC3, hsa-miR- 1304-5p, and hsa-miR-3913-5p if the step of detecting the presence of the miRNA selected from the group consisting of hsa-miR-AMC 1, hsa-miR-AMC2, hsa-miR-AMC3, hsa-miR- 1304-5p, and hsa-miR-3913-5p in the biospecimen of the patient results in identifying the patient as a candidate for receiving the antagomir.

11. The method of claim 10, wherein the miRNA is hsa-miR-AMC 1.

12. The method of claim 11, wherein the antagomir comprises SEQ ID NO: 7.