The use of mirnas in the diagnosis and treatment of distemper

Abstract

The invention relates to therapeutic and prognostic use based on the findings obtained as a result of investigating the effectiveness of different miRNAs on the expression levels of some genes involved in the Janus Kinase-Signal Transducer Transcription Activator (JAK-STAT) pathway in the treatment of respiratory form distemper.

Description

[0001] DESCRIPTION

[0002] THE USE OF miRNAs IN THE DIAGNOSIS AND TREATMENT OF DISTEMPER

[0003] Technical Field

[0004] The invention relates to therapeutic and prognostic use based on the findings obtained as a result of investigating the effectiveness of different miRNAs on the expression levels of some genes involved in the Janus Kinase-Signal Transducer Transcription Activator (JAK-STAT) pathway in the treatment of respiratory form distemper.

[0005] State of the Art

[0006] There is no specific approach to the treatment of canine distemper disease (CD), and for the specified reason, even if the animals affected by the disease are vaccinated, they can cause the death of the animal by switching to the nerve form, which is the lethal form of the disease.

[0007] While improvement is observed in some of the animals that are found to be dysthymic and start treatment, the disease progresses to nerve form in some of them. For this reason, why do some of the animals that start treatment respond to treatment, while some of them switch to the nerve form of the disease? In order to find an answer to this question, studies to investigate the immune system genetics of the host are important both up-to-date and globally.

[0008] Studies have reported that members of the JAK-STAT signaling pathway are affected by microRNA (miRNA) molecules that are not encoded by epigenetic mechanisms (Rebana et al., 2013; Kohanbash and Okada, 2012). In addition, it has been shown that viral infection can induce miRNA expression in the host organism (Li and Shi 2013), that miRNAs expressed in immune system cells target proteins in inflammatory regulation and contribute to shaping the inflammatory response (O'Connell et al., 2012). Studies have shown that miR-132 and miR- 155 are effective in different biological pathways (Ni et al., 2014; Song et al., 2019; Persson 2013; Kutty et al., 2010). In studies conducted for this purpose, it has been reported that miR- 155, one of the most researched miRNAs, provides the control of natural and acquired immune system signaling pathways (Anlar and Ba§aran, 2019) and the release of pro- inflammatory cytokines (Fabbri et al., 2012). Song et al. (2019) reported that miR-132 expression activates the JAK-STAT signaling pathway, while Chiang et al. (2013) reported that miR-132 suppresses viral replication. However, in studies conducted, it is thought that scientific information to be obtained about the Janus Kinase-Signal Transducer Transcription Activator (JAK-STAT) signaling pathway and miRNAs that can act on this pathway may contribute to the clinical level in basic immunology (Villarino, 2017). The vast majority of studies on CD disease are on phylogenetic analyses of the disease-causing Canine distemper virus (CDV) (Anis et al., 2018; Bhatt et al., 2019). Other studies on CD disease are microarray studies conducted at the in vitro level and using the tissues of animals that have died from the disease (Klotz and Gerhauser, 2019).

[0009] In the scans conducted for the JAK-STAT signaling pathway and the investigated miRNAs, 1 article and 2 patent information are noteworthy.

[0010] In the article of Chen et al. (2022), which is one of the articles that may conflict with the patent presented, they worked at the transcriptome level in the mink lung cell line infected with CDV. Chen et al., (2022) article investigated various signaling pathways associated with the immune system, including the JAK-STAT and NF-Kfl signaling pathways that are effective in the immune system. At the end of the study, it was determined that mir-140-5p and mir- 378-12 have a regulatory effect in the NF-Kfl signaling pathway, and mir-425-2, mir-139-4, mir-140-6, mir-145-3, mir-140-5p and mir-204-2 have a regulatory effect on the JAK-STAT signaling pathway.

[0011] In light of the literature and patent information summarized above, there are no prognostic and therapeutic studies on the molecular mechanisms of immunity in the host body in the presence of CDV, especially in the respiratory form of the disease, and epigenetic factors affecting these molecular mechanisms in animals infected with distemper disease.

[0012] Descriptions of the Figures

[0013] Figure 1. Gene expression changes between Oth, 24th and 48th hours

[0014] Brief Description of the Invention

[0015] The invention relates to the therapeutic use of miR-155 for canine distemper disease (CD). At the same time, it has been determined that it may be used as a prognostic parameter to predict whether a neural form will occur in dogs with CD if miR-132 is downregulated in the respiratory form of CD. Detailed Description of the Invention

[0016] There is no specific treatment for canine distemper disease (CD) in animals other than supportive treatment. Through the invention, it has been determined that the CD has the potential to form a Tyrosine Kinase 2 and Janus Kinase 1 (JAK1-TYK2) complex and that the CD can block the JAK-STAT signaling cascade. In line with the results obtained, it was seen that the changes in the expression levels of JAK1, JAK2, TYK2, miR155 and miR-132, which are involved in the biological process of the formation of the immune response in the Oh and 24h and 48h groups of the treatment against the zzzorZ>z7 / zvzrz / 5-induced distemper disease in dogs, are effective in the effectiveness of the supportive treatment applied against CD.

[0017] Animal material

[0018] The study was conducted with the permission of the Ethics Committee of the Local Ethics Committee of Animal Experiments of Erciyes University (HADYEK) (03.02.2021 / 21-29). The control group of the study consisted of 8 dogs with a large size and a healthy clinical and parasitological examination. The sick animals investigated in the study were brought to the clinics with complaints of diarrhea, vomiting, cough, fever, mucopurulent discharge in the eyes and nose, and Asan Easy Test Distemper®, Canine Distemper Virus Antigen (CDV aAg) Test (022321, Asan Pharmaceutical Co. Ltd, Korea), a total of 8 dogs, three females and 5 males, of different breeds, 2-4 months old, unvaccinated, large in size, diagnosed with CD with the rapid test kit were used. Viral positivity was confirmed by genome determination of Canine Distemper Virus (CDV) by RT-PCR method in 8 samples included in the study.

[0019] CDV diagnosis by Reverse Transcriptase-PCR

[0020] CDV was diagnosed by the RT-PCR method from RNA samples obtained from dogs. Following RNA isolation from blood samples, RT-PCR was performed in the QiagenThermal Cycler device using the Qiagen Onestep Reverse Transcriptase-PCR kit (Qiagen, Hilden, Germany) in accordance with the method reported by Frisk et al. (1999) to determine the presence of the nucleocapsid gene in the CDV genome.

[0021] Collection of blood samples and hematological examination

[0022] Blood samples were collected from the vena cephalica antebrachii of the dogs included in the study into EDTA tubes in the control, pre-treatment (Oth hour), 24th hour (24h) and 48th hour (48h) groups. Complete blood count was performed using a hemogram device (MindrayBC- 2800 Vet®, China) from blood samples taken into tubes with EDTA. Treatment protocol

[0023] Supportive treatment protocol was applied to dogs diagnosed with CD positive. The treatment steps applied are fluid therapy, antibiotic, immune modulator, and vomiting inhibitor applications, respectively. In fluid treatment, the amount of fluid required was calculated by considering the hematocrit value, rehydration was provided with lactated ringer intravenous (IV) with 5% dextrose, 0.09% NaCl, and applied until the animal started to eat itself (7 days) (§ahal et al., 1994; Ocal et al., 2006). In order to prevent secondary infections, 30 mg / kg cefazolin Cefazolin® (Eqizolin®, Turn Ekip flap, Istanbul, Tiirkiye) was administered at 12- hour intervals; 10mg / 5 ml P-glucan (Imuneks®, ImuneksFarma, Istanbul, Tiirkiye) as an immunomodulator twice a day; 25-500 mg / kg Vit C (Maxivit C®, baVET, Istanbul, Tiirkiye); 1.5-2ml vit B1-B6 (Nervit®, Vetas, Istanbul, Tiirkiye); 2.2-4.4mg / kg B12 (Butafan®, baVET, Istanbul, Tiirkiye) was administered. In addition, in animals with vomiting, 0.5 mg / kg Metoclopramide (Metpamid®, Sifar, Istanbul, Tiirkiye) and 2 mg / kg ulcuran (Ranitidine®, Levent, Istanbul, Tiirkiye) were administered as vomit inhibitors and EE receptor blockers, respectively (Daldaban et al. 2021).

[0024] RNA Isolation

[0025] RNA isolation was performed on the same day following blood collection. Total RNA isolation was performed manually (Qiagen, Hilden, Germany) with Trizol from whole blood samples. In order to prevent DNA contamination that may be found in the obtained RNA samples, DNAase was applied with the Invitrogen DNA-free DNA removal kit (Invitrogen, Thermo Fisher Scientific, Carlsbad, US). The concentrations of the RNAs obtained in the next stage were determined and kept at -80°C until the working day.

[0026] Gene expression analysis by RT-qPCR mRNA RT-qPCR c-DNA synthesis was performed in the Qiagen thermal cycler (Qiagen SensoQuest, Hilden, Germany) device according to the Transcriptor First Strand c-DNA Synthesis Kit (Roche Ltd, Mannheim, Germany) protocol. The synthesized c-DNA samples were diluted 1 / 4 with Nuclease Free Water (NFW). The expression stage of the study was studied with the Fast Start SYBR Green Master Mix (Roche Ltd, Mannheim, Germany) kit in triplicate in the LightCycler Nano (Roche Ltd, Mannheim, Germany) device. Melting curve analysis was performed to prevent nonspecific radiations that may occur in the study. The data of the target genes were normalized with housekeeping genes. The 2-AACt formula was applied to the normalized data and made suitable for statistical analysis by using the raw data obtained (Livak and Schmittgen, 2001). The primary sequences used in the study are given in Table 1.

[0027] When viral diseases such as CD disease come into contact with the organism, natural and acquired immune mechanisms activate the immune system of the host organism (Maillard, 2013). In organisms infected with viruses, the JAK-STAT signaling pathway is mostly targeted to prevent or avoid the IFN-based immune response (Cheee and Roizman, 2004). The Janus Kinase-Signal Transducer Transcription Activator (JAK-STAT) signaling pathway, which has been defined as a cytokine-induced signaling pathway in mammals, plays an active role in many biological mechanisms such as hematopoietic development and differentiation, embryonic development, innate and acquired immune system (Raftery and Stevenson, 2017). The JAK and STAT signaling pathway activators are the molecules that make up the JAK- STAT signaling pathway, which is necessary for immune regulation (Traves et al., 2021). In mammals, the JAK gene family has four members, JAK1, JAK2, JAK3 and tyrosine kinase 2 (TYK2) while the STAT gene family consists of seven members, STAT1, STAT2, STAT3, STAT4, STAT5A, STAT5B and STAT6 (Samuel, 2001). Members of the JAK-STA T signaling pathway have been reported to be affected by microRNA molecules (miRNA) that are not encoded by epigenetic mechanisms (Rebana et al., 2013; Kohanbash and Okada, 2012). Viral infection can induce miRNA expressions in the host organism (Li and Shi 2013), and it has been determined that miRNAs expressed in immune system cells contribute to shaping the inflammatory response by targeting proteins in inflammatory regulation (O'Connell et al., 2012). Studies have shown that miR-132 and miR-155 are effective in different biological pathways (Ni et al., 2014; Song et al., 2019; Persson 2013; Kutty et al., 2010). -In studies conducted for this purpose, it has been reported that miR-155, one of the most researched miRNAs, provides the control of natural and acquired immune system signaling pathways (Anlar and Ba§aran, 2019) and the release of pro-inflammatory cytokines (Fabbri et al., 2012). Song et al. (2019) reported that miR-132 expression activates the JAK-STAT signaling pathway, while Chiang et al. (2013) reported that miR-132 suppresses viral replication. Table 1. Primary sequences of genes studied in RT-qPCR analysis miRNA RT-qPCR

[0028] The primary sequences of miRNAs (miR-132 and miR-155) investigated in the study were obtained from miRBase (Table 2). c-DNA synthesis was performed for miR-132 and miR- 155 investigated. c-DNA synthesis was performed in the Qiagen thermal cycler (Qiagen SensoQuest, Hilden, Germany) device according to the Mir-X™ miRNA First-Strand Synthesis Kit (Takara Bio, Japan, Kat No: 638313) protocol with c-DNA synthesis RNA samples. c-DNAs were diluted with nuclease free water (NFW) at a rate of 14 and studied with the Mir-X™ miRNA qRT-PCR TB Green® Kit (Takara Bio, Japan, Kat No: 638316) kit in triplicate in the LightCycler Nano (Roche Ltd, Mannheim, Germany) device. Melting curve analysis was performed to prevent nonspecific radiations that may occur in the study. The data of the target genes were normalized with housekeeping genes. The 2-AACt formula was applied to the normalized data and made suitable for statistical analysis by using the raw data obtained (Livak and Schmittgen, 2001).

[0029] Table 2. miRNA primary sequences studied in RT-qPCR analysis Statistical analyses

[0030] The suitability of the data for normal distribution was evaluated by skewness-kurtosis, histogram, q-q plot graphs and Shapiro-Wilk test. The differences between the control and Oh groups in terms of gene expression levels and genotypic and hematological parameters were tested with an independent two-sample t-test. Differences in expression parameters between the repeated measurements of the groups at different periods of treatment (Oh, 24h and 48h) were tested with ANOVA in repeated measurements. However, the relationships between gene expression levels and hematology parameters and the relationships between gene expression levels were determined by the Pearson correlation coefficient. IBM SPSS 14.01 program was used in the statistical analysis of the data and the significance level was considered p<0.05.

[0031] At the end of the study, it was thought that miR-155 could be used therapeutically for CDV in dogs with distemper and miR-132 in the respiratory form of CD could be an important prognostic parameter to predict whether a neural form will occur in dogs with CD. In line with the results obtained, it is thought that the changes in the expression levels of JAK1, JAK2, TYK2, miR155 and miR-132, which are involved in the biological process of immune response in pre-treatment Oh and 24h and 48h groups of treatment against morbillivirus- induced distemper disease in dogs, may be effective in the effectiveness of supportive treatment against CDV.

[0032] In the study, three females-five males in the distemper group and three females-five males 2-4 months old unvaccinated large dog breeds were used in the control group. The dogs in the distemper group had mucopurulent discharge, cough, dehydration, body temperature between 38.5-40.3°C (average 39.3°C), diarrhea in four dogs and vomiting in one dog. The degree of dehydration was recorded as mild in two dogs, moderate in four dogs, and severe in two dogs.

[0033] As a result of the treatment applied to the animals in the distemper group for seven days, it was determined that their dehydration improved, cough, nasal and eye discharge disappeared, their interest in the environment increased, their body temperatures decreased to normal limits, and their vomiting and diarrhea stopped.

[0034] In the analysis performed between the control group and the CDV group at the Oth hour, a significant difference (p<0.05) was found between the expression levels of JAK1, JAK2, TYK2, miR-155 and miR-132 (Table 3). Table 3: Gene expression comparisons of the control group and the CDV group (Oh hour)

[0035] As a result of the analysis performed between the JAK1, JAK2, TYK2, STAT1, STAT2, miR- 132 and miR-155 gene expression levels at the Oth, 24th and 48th hours in the CDV group, a statistically significant difference (p<0.05) was found in the JAK2 and TYK2 gene expression levels (Table 4).

[0036] Table 4: Comparison of gene expression levels between Oh, 24h and 48h in the CDV group

[0037]

[0038] Multiple comparison tests were performed to investigate the time-dependent changes in the expression levels of the studied miRNAs and genes at the Oth, 24th and 48th hours. In the analyses, it was observed that there was a statistically significant difference (p<0.05) between the Oth and 48th hours in the CDV group in the JAK2 gene and between the 24th and 48th hours in the TYK2 gene (Figure 1).

[0039] According to the correlation coefficients between the expression levels of the studied genes, it was determined that there was a positive correlation between STAT1 and JAK2\ a positive correlation between miR132 and STAT2,' and a negative correlation between miR155 and JAK2 and STAT1 in the control group. However, according to the correlation coefficients between the expression levels of the studied genes, it was determined that there was a positive correlation between STAT1 and JAK2 and a positive correlation between miR-132 and JAK1 at the Oth hour of the CDV group (Table 5). Table 5. Correlations of the expression values of the genes between the control and the CDV group (Oth hour) In this study, the relationship between the expression levels of the JAK1, JAK2, TYK2, STAT1 and STAT2 genes involved in the activation of the immune system and the expression levels of miR-132 and miR-155, which play a role in the formation of the immune response against the canine distemper virus (CDV), was investigated. The animal material of the study consisted of healthy large dogs (control, n=8) and 2-4 months old, unvaccinated, large dogs (CDV, n=8) diagnosed with distemper. Blood samples were collected once from the control group included in the study and at the pre-treatment (Oh), post-treatment 24th and 48th hours from the CDV group. Expression analysis of the miR-132, miR-155, JAK1, JAK2, TYK2, STAT1 and STAT2 genes taken from the blood samples was performed by the RT-qPCR method. A statistically significant difference (p<0.05) was found between the JAK1, JAK2, TYK2, miR-155 and miR-132 expression levels (p<0.05) between the control group and the CDV group at Oh and between the JAK2 and TYK2 gene expression levels at Oh, 24h and 48h in the CDV group. In the multiple comparison analyses, it was observed that there was a statistically significant difference (p<0.05) between the Oh and 48h hours of the JAK2 gene expression level and between the 24h and 48h hours of the TYK2 gene expression level. However, according to the correlation coefficients, it was determined that there was a positive correlation between the Oh group STAT1 and JAK2 and a positive correlation between miR- 132 and JAKE

[0040] As a result, it has been determined that CDV has the potential to form a TYK2 and JAK1 complex and that CDV can block the JAK-STAT signaling cascade. It is thought that miR-155 may be a biomarker for the distemper and that the upregulation occurring in the miR155 gene expression is supported in the presence of P-glucan. In addition, it was thought that the downregulation of miR-132 in the respiratory form of CDV may be an important prognostic parameter to predict whether a neural form will occur in dogs with CDV. In line with the results obtained, it is thought that the changes in the expression levels of JAKL JAK2, TYK2, miR155 and miR-132, which are involved in the biological process of immune response formation in dogs at pre-treatment Oh and 24h and 48h hours of treatment against morbillivirus-induced distemper disease, may guide the effectiveness of supportive therapy against CDV.

Claims

CLAIMS1. A biomarker for the detection of canine distemper disease or its level, characterized in comprising the following sequences:

2. The biomarker according to claim 1, characterized in that the sequenceis used for the detection of canine distemper disease.

3. The biomarker according to claim 1, characterized in that the sequenceis used for the detection of whether canine distemper disease has passed into the neural form.

4. A therapeutic or prognostic composition, characterized in comprising a biomarker according to claim 1.

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