Method for treating sequela of RNA rivus infection

Abstract

The present invention provides a method for treating a sequela of an infection by a SARS-CoV-2 virus, the method including administering an inhibitor of an RNA-dependent RNA polymerase (RdRp) of the virus to a patient who needs the treatment. Also provided are: a method for screening for a therapeutic agent for a sequela of an infection by the virus; and a model mouse useful for the method.

Description

Method for treating sequelae of RNA virus infection

[0001] The present invention relates to a method for treating the aftereffects of RNA virus infection, particularly the aftereffects of infectious diseases such as the novel coronavirus. More specifically, the present invention provides a novel treatment method for the aftereffects of infectious diseases such as the novel coronavirus using an RNA-dependent RNA polymerase (RdRp) inhibitor, and a transgenic conditional knock-in mouse designed by genetic modification to induce the expression of Nsp12, a protein of the RNA-dependent RNA polymerase of the novel coronavirus (SARS-CoV-2).

[0002] Patients with the aftereffects of COVID-19 infection have reported severe fatigue, myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS)-like symptoms, post-exertional fatigue, and postural orthostatic tachycardia syndrome (POTS). 1-12 These symptoms are often accompanied by other abnormalities that can lead to heart failure. 1-4、6-14 Similar aftereffects of viral infections (such as severe fatigue, ME / CFS, and POTS) can occur not only as a result of COVID-19 infection, but also as a result of RNA virus infections such as Ebola virus, dengue virus, and influenza virus. 3 In patients with sequelae of COVID-19 infection, significant fluctuations are observed not only in immune responses but also in cytokine and hormone levels, with women experiencing the disease more frequently than men. 15-20 Furthermore, SARS-CoV-2 RNA was detected in this patient even two years after the onset of symptoms, suggesting that the formation of a long-term viral reservoir may be a cause of the sequelae of COVID-19 infection. 21 In order to develop effective therapeutic agents for patients with the aftereffects of COVID-19 infection, it is essential to clarify how the SARS-CoV-2 virus damages host cells in vivo and identify the viral factors that cause this damage. Recently, it has been reported that patients with acute COVID-19 infection and its aftereffects show signs of mitochondrial dysfunction. 9,22-25It has been reported that increased expression of miRNA-2392, which inhibits oxidative phosphorylation (OXPHOS), is involved in this mitochondrial dysfunction. 22,24 Indeed, it was demonstrated that SARS-CoV-2-infected mice exhibited reduced levels of mitochondrial gene expression in the heart and kidney. 26 Additionally, in vitro studies have shown that the SARS-CoV-2 M, N, Nsp6, ORF9b, and ORF10 proteins directly bind to mitochondrial proteins. 27-29 However, the mechanism by which SARS-CoV-2 virus infection causes mitochondrial damage in host cells in vivo remains unknown, and no SARS-CoV-2 viral factors have been identified as targets for preventing or treating mitochondrial dysfunction.

[0003] The present invention aims to provide a new treatment method for the aftereffects of infectious diseases such as the novel coronavirus using an RNA-dependent RNA polymerase inhibitor, and to provide a model animal useful for screening pharmaceuticals for treating the aftereffects.

[0004] The viral factors responsible for the onset of the sequelae of COVID-19 infection are unknown. Here, we describe a conditional knock-in (cKI) mouse strain we created that expresses Nsp12, the active subunit of the RNA-dependent RNA polymerase (RdRp) of the novel coronavirus (SARS-CoV-2).

[0005] The integrated stress response (ISR) is a host defense system that prevents translation of foreign RNA viruses. 30In vitro and in vivo, Nsp12 translation in host cells was suppressed by this ISR mechanism, and induction of Nsp12 expression required the combination of an ISR inhibitor. We demonstrated a relationship between this induction of Nsp12 expression and mitochondrial dysfunction in vivo. First, in vitro, induction of Nsp12 expression in primary lung epithelial cells from Nsp12 cKI mice suppressed mitochondrial function. Furthermore, in vivo, expression of Nsp12 in mouse lungs did not result in pneumonia, pulmonary thrombosis, or pulmonary fibrosis, but induction of Nsp12 expression caused mitochondrial abnormalities in the heart of Nsp12 cKI mice both early and long-term after induction.

[0006] Intravenous administration of EIDD-2801 (molnupiravir), an RNA-dependent RNA polymerase (RdRp) inhibitor, restored mitochondrial function in cardiomyocytes of Nsp12 cKI mice. These findings suggest that SARS-CoV-2 RdRp activity in the lungs can cause cardiac mitochondrial dysfunction, resulting in a phenotype similar to that observed in the aftereffects of COVID-19 infection in humans. Therefore, therapeutic targeting of SARS-CoV-2 RdRp offers a novel approach to prevent or alleviate symptoms of severe fatigue, myalgic encephalomyelitis / chronic fatigue syndrome (ME / CFS), postural orthostatic tachycardia syndrome (POTS), and heart failure caused by mitochondrial dysfunction in patients with COVID-19 and other aftereffects.

[0007] As a result of extensive research, the inventors discovered that RNA-dependent RNA polymerase (RdRp) inhibitors, which are used as therapeutic agents for the novel coronavirus, Ebola virus, influenza virus, etc., are effective in treating the aftereffects of COVID-19 by restoring mitochondrial function, thereby completing the present invention. The present invention was also completed by generating conditional knock-in (cKI) mice engineered through genetic modification to induce the expression of Nsp12, a protein of the novel coronavirus (SARS-CoV-2) RNA-dependent RNA polymerase (RdRp).

[0008] The objects of the present invention are achieved by the following (1) to (16): (1) A method for treating sequelae of an RNA virus infection, comprising administering a therapeutically effective amount of an RNA-dependent RNA polymerase (RdRp) inhibitor to a subject in need thereof, including a human. (2) The method according to (1), wherein the sequelae are sequelae of infection with a novel coronavirus, Ebola virus, dengue virus, influenza virus, or the like. (3) The method according to (2), wherein the sequelae are sequelae of infection with a novel coronavirus. (4) The method according to (3), wherein the symptoms of the sequelae are caused by organ damage due to mitochondrial dysfunction. (5) The method according to (4), wherein the organ damage is organ damage in the lungs and / or heart. (6) The method according to any of (1) to (5), wherein the RdRp inhibitor is selected from the group consisting of molnupiravir, remdesivir, favipiravir, and ribavirin. (7) A pharmaceutical composition for treating sequelae of a viral infection in a subject in need thereof, including a human, comprising a therapeutically effective amount of an RNA-dependent RNA polymerase (RdRp) inhibitor and a pharmaceutically acceptable excipient. (8) The pharmaceutical composition according to (7), wherein the sequelae are sequelae of infection with a novel coronavirus, Ebola virus, dengue virus, influenza virus, or the like. (9) The pharmaceutical composition according to (8), wherein the sequelae are sequelae of infection with a novel coronavirus. (10) The pharmaceutical composition according to (9), wherein the symptoms of the sequelae are caused by organ damage due to mitochondrial dysfunction. (11) The pharmaceutical composition according to (10), wherein the organ damage is organ damage in the lungs and / or heart. (12) The pharmaceutical composition according to any of (7) to (11), wherein the RdRp inhibitor is selected from the group consisting of molnupiravir, remdesivir, favipiravir, and ribavirin. (13) Transgenic conditional knock-in mice engineered by genetic modification to induce the expression of the novel coronavirus RNA-dependent RNA polymerase (RdRp) protein Nsp12.(14) A lung-specific transgenic conditional knock-in mouse designed by genetic modification to induce expression of the novel coronavirus RNA-dependent RNA polymerase (RdRp) protein Nsp12. (15) A method for screening therapeutic agents for the aftereffects of novel coronavirus infection using the knock-in mouse described in (13). (16) A method for screening therapeutic agents for the aftereffects of novel coronavirus infection using the knock-in mouse described in (14).

[0009] The RNA-dependent RNA polymerase (RdRp) inhibitor used in the present invention improves mitochondrial function that is impaired as a sequela of infectious diseases such as the novel coronavirus, and is useful for treating symptoms caused by such impairment. The same applies to transgenic conditional knock-in mice designed by genetic modification to express the RNA-dependent RNA polymerase (RdRp) protein Nsp12 of the novel coronavirus (SARS-CoV-2) used in the present invention, and screening methods using such mice.

[0010] Figure 1 shows generation of Rosa26-LSL-Nsp12 conditional knock-in (cKI) mice. Figure 2 shows PCR analysis to confirm homologous recombination in ES cells. Figure 3 shows Southern blot hybridization to confirm homologous recombination in ES cells. Figure 4 shows PCR analysis of cross products of heterozygous Rosa26-LSL-Nsp12 cKI mice. Figure 5 shows the results of the analysis of ... T2 1 is a diagram showing mitochondrial dysfunction in primary lung epithelial cells derived from Nsp12 mice and the ameliorating effect of an RdRp inhibitor (Example 2). T21 is a diagram showing mitochondrial dysfunction in primary lung epithelial cells derived from Nsp12 mice and the ameliorating effect of EIDD-2801 (molnupiravir) (Example 3). It is a diagram of fluorescent immunostaining images showing mitochondrial damage in mouse hearts caused by induction of Nsp12 expression and the therapeutic effect of EIDD-2801 (molnupiravir) (Example 4). It is an electron microscope image showing mitochondrial damage in mouse hearts caused by induction of Nsp12 expression and the therapeutic effect of EIDD-2801 (molnupiravir) (Example 5). It is an electron microscope image showing chronic mitochondrial damage in mouse hearts caused by induction of Nsp12 expression and the therapeutic effect of EIDD-2801 (molnupiravir) (Example 6).

[0011] (1) RNA-Dependent RNA Polymerase Inhibitors In the present invention, RNA-dependent RNA polymerase (RdRp) inhibitors can be used in the form of conventional pharmaceuticals for oral or parenteral administration, such as intravenous infusion, as pharmaceuticals for treating the aftereffects of COVID-19 infection or at least one symptom thereof. Specific examples of RdRp inhibitors include molnupiravir, remdesivir, favipiravir, and ribavirin. These compounds include their isomers, pharmaceutically acceptable salts, solvates, hydrates, prodrugs, esters, or active metabolites (e.g., GS-441524, an active metabolite of remdesivir; T-705 ribofuranose, an active metabolite of favipiravir; and EIDD-1931, an active metabolite of molnupiravir). Pharmaceuticals for oral administration include solid formulations such as tablets, granules, powders, and capsules, as well as liquid formulations such as syrups. These formulations can be prepared using conventional methods. Solid preparations can be prepared using conventional pharmaceutical carriers such as lactose, starches such as cornstarch, microcrystalline cellulose, hydroxypropyl cellulose, carboxymethylcellulose calcium, talc, magnesium stearate, etc. Capsules can be prepared by encapsulating the granules or powders prepared in this manner. Syrups can be prepared by dissolving or suspending the compound in an aqueous solution containing sucrose, carboxymethylcellulose, etc. Pharmaceuticals for parenteral administration include preparations for injections such as intravenous drips. Injectable preparations can also be prepared by conventional methods and, if necessary, can contain tonicity adjusters (e.g., mannitol, sodium chloride, glucose, sorbitol, glycerol, xylitol, fructose, maltose, mannose), stabilizers (e.g., sodium sulfite, albumin), and preservatives (e.g., benzyl alcohol, methyl parahydroxybenzoate).

[0012] (2) Transgenic conditional knock-in mice. The present invention provides transgenic conditional knock-in mice and lung-specific transgenic conditional knock-in mice engineered by genetic modification to express the Nsp12 protein of the RNA-dependent RNA polymerase (RdRp) of the novel coronavirus (SARS-CoV-2). SARS-CoV-2 is a positive-strand RNA virus with a genomic RNA of approximately 30 kbp. 31 In infected host cells, the SARS-CoV-2 viral RNA genome is translated and replicated using both viral and host factors. The SARS-CoV-2 RNA-dependent RNA polymerase (RdRp) Nsp12 is a crucial viral factor with enzymatic activity required for SARS-CoV-2 RNA genome replication. To analyze the effects of SARS-CoV-2 RdRp Nsp12 itself on host cells in vivo, we established a Rosa26-LSL-Nsp12 conditional knock-in (cKI) mouse strain in which a LoxP-Stop-LoxP (LSL) sequence and an Nsp12-ires (internal ribosome entry site)-GFP (green fluorescent protein) cDNA were placed downstream of the Rosa26 locus (Figure 1). When the mice were 3 to 5 weeks old, TAT-Cre, a recombinant Cre recombinase fused with a cell-permeable amino acid sequence, was administered intravenously for 8 days to induce expression of the Nsp12 protein by deleting the LSL sequence. These mutants were named "Nsp12 mice." Furthermore, Rosa26-LSL-Nsp12 mice and alveolar type II (AT2) stem cell-specific Sftpc-CreER mice were also developed. T2 Mice were bred and transfected with Sftpc-CreER T2 By generating Rosa26-LSL-Nsp12 mice, we established a lineage in which Nsp12 expression is lung-specific. When the mice were 3 to 5 weeks old, we administered (Z)-4-hydroxytamoxifen (4-OHT) intravenously for 8 days to induce Nsp12 protein expression in lung tissue by deleting the LSL sequence. The resulting mutant was a "CreER" mutant. T2;Nsp12 mouse.

[0013] Example 1: Creation of transgenic conditional knock-in mice and lung-specific transgenic conditional knock-in mice engineered by genetic modification to express the novel coronavirus RNA-dependent RNA polymerase (RdRp) protein Nsp12;

[0014] Establishment of Rosa26-LSL-Nsp12 Conditional Knock-in (cKI) Mice. The pLVX-EF1α-SARS-CoV-2-nsp12-2xstrep-IRES-Puro plasmid (Addgene #141378) was purchased from Addgene. To establish Rosa26 cKI mice conditionally expressing SARS-CoV-2 Nsp12, a loxP-flanked STOP cassette (LSL), a neomycin resistance gene, SARS-CoV-2 Nsp12 cDNA, an ires (internal ribosome entry site), and GFP (green fluorescent protein) were inserted into the mouse Gt(ROSA)26Sor locus (Figure 1). To construct the targeting vector, a 3.3-kb upstream DNA fragment and a 4.3-kb downstream DNA fragment associated with the unique XbaI site in intron 1 of the Gt(ROSA)26Sor locus were transfected into RENKA ES cells. 32 The genomic fragments were amplified by PCR from the genomic DNA of the targeting vector and used as homologous arms for the targeting vector. These amplified genomic fragments contained the MC1_DTA cassette (polyoma enhancer / Herpes simplex virus thymidine kinase promoter-driven diphtheria toxin A gene) as a negative selection marker, a splice acceptor sequence, and a stuffer sequence. 33The expression unit was subcloned into a plasmid containing an LSL cassette containing CAT (chloramphenicol acetyltransferase) cDNA as a positive selectable marker. A PGK neo cassette (phosphoglycerate kinase I promoter-driven neomycin resistance gene) flanked by FRT was inserted downstream of the CAT sequence as a positive selectable marker. To construct the expression unit, the SARS-CoV-2 Nsp12 gene coding sequence and ires-GFP were amplified by PCR and subcloned, along with a bGH (bovine growth factor) poly(A) signal, into the targeting vector downstream of the second loxP site of the STOP cassette. The resulting targeting vector contained MC1_DTA, a 3.3 kb 5' homology arm, a splice acceptor, a first loxP site, CAT cDNA, poly(A), frt-flanked PGK neo cassette, a second loxP site, Nsp12-ires-GFP, bGH poly(A), and a 4.3 kb 3' homology arm. The Nsp12 expression vector was linearized and introduced into RENKA ES cells (C57BL / 6N) by electroporation. After selection using Geneticin®, resistant clones were isolated and DNA was screened for homologous recombination by PCR using the following primer set (rosa_F2731: 5'-CCA TGC TGG AAG GAT TGG AAC TAT GC-3' and SA-R3: 5'-AGG ATA AGT ATG ACA TCA AGG-3'). PCR-positive ES clones were expanded, and DNA isolated from each clone was verified by PCR amplification using the following primer sets: rosa_F2731 and SA-R3 for 5' amplification, and neo100: 5'-AGG TGA GAT GAC AGG AGA TC-3' and rosa_R10578: 5'-AAG CTT ACC ATC AAC CTT ATA GTA CAC-3' for 3' amplification (Fig. 2a, b). Homologous recombination in these clones was also confirmed by genomic Southern blot hybridization using the neomycin resistance gene and GFP gene as probes (Fig. 3a, b). Homologous recombination ES cell clones were mixed with ICR 8-cell embryos to generate chimeric mice.By crossing chimeric mice with a high RENKA background contribution with C57BL / 6N mice, F1 heterozygous mice showing germline transmission were obtained. The targeted allele was identified by PCR using the primer set rosa_F2731 and SA-R3.

[0015] Genotyping of Rosa26-LSL-Nsp12 cKI mice Heterozygous Rosa26-LSL-Nsp12 fl / - Mice were bred to homozygous Rosa26-LSL-Nsp12 fl / fl Mice were obtained and genotyped using the following primer sets: rosa-F5982: 5'-TTT GGA GGC AGG AAG CAC TTG CTC TCC-3'; rosa-R6262: 5'-ACA CAC CAG GTT AGC CTT TAA GCC CTG-3'; GFP-F649: 5'-GAT CAC ATG GTC CTG GAG TTC-3'. For PCR analysis, genomic DNA was subjected to 35 cycles of 94°C for 30 seconds, 63°C for 30 seconds, and 72°C for 30 seconds, amplifying a 441-bp product from the Rosa26-LSL-Nsp12 allele and a 281-bp product from the wild-type allele (Fig. 4). PCR was performed using a KOD FX (Toyobo, Osaka, Japan).

[0016] CreER T2 To investigate the effect of lung-specific expression of the SARS-CoV-2 Nsp12 gene, we used Sftpc-CreER conditional cKI mice. T2 Transgenic mice (JAX database #0028054, Jackson Laboratory, Bar Harbor, ME, USA) were purchased. Sftpc-CreER T2 Transgenic mice and Rosa26-LSL-Nsp12 fl / - Mice were bred to express lung-specific CreER T2 ;Nsp12 mice were established.

[0017] Example 2 CreER T2Mitochondrial dysfunction in primary lung epithelial cells derived from Nsp12 mice and the ameliorative effect of RdRp inhibitors 1) CreER T2 Isolation of primary lung epithelial cells from Nsp12 mice T2 Bilateral lungs removed from Nsp12 mice were digested with Dri Tumor & Tissue Dissociation Reagent (BD Biosciences, Franklin Lake, NJ, USA) and then stained with mouse anti-CD31 antibody (Biolegend, San Diego, CA, USA), mouse anti-CD45 antibody (Biolegend), mouse anti-CD16 / 32 antibody (Biolegend), and BD iMag TM Primary lung epithelial cells were isolated by negative selection using Streptavidin Particle Plus (BD Biosciences).

[0018] 2) CreER T2 Culture of primary lung epithelial cells from Nsp12 mice (PneumaCult) TM EX Plus medium (Stemcell Technologies, Vancouver, Canada) was supplemented with 5 μM SB203580 (LC Laboratories, Woburn, MA, USA), 1 μM CHIR-99021 (FOCUS Biomolecules, Premath Meeting, PA, USA), 10 μM Y-27632·2HCl (Selleckchem, Houston, TX, USA), 5 μM DMH1 (Selleckchem), 5 μM A83-01 (Tocris Bioscience, Bristol, UK), 5 ng / ml Wnt3a (R&D Systems, Minneapolis, MN, USA), 50 ng / ml R-Spondin 3 (R&D Systems), 20 ng / ml EGF (Stemcell Technologies), 10 ng / ml bFGF (Stemcell Technologies), and 50 ng / ml Noggin (Fujifilm Wako Pure Chemical Industries, Osaka, Japan) and incubated at 37°C with CreER. T2 Nsp12-derived mouse primary lung cells were cultured.

[0019] 3) Effect of RdRp inhibitors on mitochondrial function in primary lung epithelial cells. CreER cells were pretreated with 1 μM ISRIB (trans isomer, Selleckchem). T2 Primary lung epithelial cells from Nsp12 mice were cultured in PneumaCult media supplemented with 1 μM 4-OHT (Abcam, Cambridge, UK) and 1 μM ISRIB. TM Nsp12 expression was induced by culturing the cells in EX Plus medium for two days. In parallel, RdRp inhibitors were added to the culture medium and cultured for two days to examine the effect of the RdRp inhibitors on the Nsp12-mediated reduction of mitochondrial function in these cells. The RdRp inhibitors added were as follows: GS-441524, T-705 ribofuranose, and EIDD-1931, which are activated compounds produced by the metabolism of remdesivir, favipiravir, and molnupiravir, respectively. They were 10 μM GS-441524 (MedChemExpress, Mammoth Junction, NJ, USA), 10 μM T-705 ribofuranose (Toronto Research Chemicals Inc., Toronto, Canada), and 10 μM EIDD-1931 (MedChemExpress).

[0020] The primary lung epithelial cells were seeded onto poly-L-lysine (Merck, St. Louis, MO, USA)-coated glass slides and incubated with 50 nM MitoTracker. TM PneumaCult supplemented with DeepRed FM (Thermo Fisher Scientific, Waltham, MA, USA) TMAfter incubation in EX Plus medium at 37°C for 2 hours, the cells were fixed with 4% PFA (paraformaldehyde). Fixed cells were stained with rabbit anti-SARS-CoV-2 RNA-dependent RNA polymerase Nsp12 antibody (Cell Signaling Technologies, Danvers, MA, USA), AlexaFluor 488-conjugated donkey anti-rabbit IgG secondary antibody (Molecular Probes®, Thermo Fisher Scientific), and DNA marker DAPI (Dojindo Laboratories, Mashiki, Kumamoto, Japan). Fluorescent images were then captured using a Stellaris 5 confocal laser scanning microscope (Leica Microsystems GmbH, Wetzlar, Germany). In this image, green indicates the rabbit anti-Nsp12 antibody, and red indicates MitoTracker. TM The blue color indicates the results of DAPI (nuclei) staining, and the white line represents the scale bar (10 μm). As shown in Figure 5, this study demonstrated that mitochondrial function was suppressed in Nsp12-expressing cells (Nsp12+) compared to non-Nsp12-expressing cells (Nsp12-). Furthermore, this reduction in mitochondrial function was restored by treatment with an RdRp inhibitor.

[0021] Example 3 CreER T2 Mitochondrial dysfunction in primary lung epithelial cells derived from Nsp12 mice and the ameliorating effect of EIDD-2801 (molnupiravir) T2 Primary lung cells derived from Nsp12 mice were co-treated with the RdRp inhibitor EIDD-2801 (molnupiravir) (10 μM, Merck, St. Louis, MO, USA) to analyze the effect of Nsp12 on mitochondrial dysfunction. As shown in Figure 6, this study demonstrated that the mitochondrial dysfunction observed in Nsp12-inducible cells (Nsp12+) was reversed by co-treatment with EIDD-2801 (molnupiravir).

[0022] Example 4 Fluorescent immunostaining images showing mitochondrial damage in mouse hearts due to induction of Nsp12 expression and the therapeutic effect of EIDD-2801 (molnupiravir) 1) In vivo Nsp12 mice and CreER T2 Induction of Nsp12 expression in Nsp12 mice. To examine the effect of inducing Nsp12 expression in the mouse lung in vivo, we used lung-specific CreER T2 Nsp12 mice and wild-type (C57BL / 6) control mice (Control) were intravenously injected with (Z)-4-hydroxytamoxifen (4-OHT, 5 mg / kg, Abcam, Cambridge, UK) dissolved in serum-free stem cell medium S-Clone SF-03 (-) (BSA-, cytokine-, penicillin / streptomycin-free, Iwai North America, Signal Hill, CA, USA) on days 0, 1, 2, 3, and 7, 8, 9, and 10. On the same days, Nsp12 mice and wild-type (C57BL / 6) control mice (Control) were intravenously injected with TAT-Cre recombinase (1,000 units / kg, Merck Millipore, Burlington, MA, USA) dissolved in SF-03 (-). To suppress the ISR in the mice and induce protein expression through Nsp12 translation, the ISR inhibitor, ISRIB (trans isomer, 5 mg / kg, Selleckchem) was dissolved in SF-03 (-) and intravenously injected on days 1 and 6. Additionally, A92 (GCN2-IN-1, 0.4 mg / kg, MedChemExpress, Mammoth Junction, NJ, USA) was dissolved in artificial gastric fluid (2.0 g NaCl, 7 ml concentrated hydrochloric acid, 3.2 g pepsin dissolved in 993 ml purified water) and orally administered on days 0, 1, 2, 3, and 7, 8, 9, 10, and 11. Therefore, to induce Nsp12 expression in vivo, CreER T2Nsp12 mice or Nsp12 mice were orally or intravenously administered 4-OHT or TAT-Cre along with the above-mentioned ISR inhibitors (ISRIB and A-92) on days 0, 1, 2, 3, and 7, 8, 9, 10, and 11. To analyze the effects of RdRp inhibitors on Nsp12, the mice were orally administered artificial gastric juice containing EIDD-2801 (10 mg / kg, Merck, St. Louis, MO, USA) on days 0, 1, 2, 3, and 7, 8, 9, 10, and 11. Wild-type C57BL / 6 mice (CLEA Japan, Tokyo, Japan) served as control mice.

[0023] 2) Fluorescent immunohistochemical staining of frozen heart sections. To prepare frozen heart sections, wild-type (C57BL / 6) mice and CreER mice were used. T2Heart tissue from Nsp12 mice was fixed in Periodate Lysine Paraformaldehyde (PLP) solution (Fujifilm Wako Pure Chemical Industries, Ltd.) by incubation on ice for 1 hour. Cryosections (10 μm thick) of the mouse heart tissue were prepared using a cryostat (7732, CM1950, Leica Microsystems GmbH, Wetzlar, Germany). The sections were washed twice with 2% BSA TBS solution and blocked for 1 hour with 2% BSA TBS solution. The sections were then incubated overnight at 4°C with mouse anti-ATP5H antibody (Thermo Fisher Scientific, Waltham, MA, USA) and rabbit anti-phospho-p62 (SQSTM1) (Ser351) antibody (MBL, Nagoya, Aichi, Japan). Next, the sections were stained with AlexaFluor 488-conjugated donkey anti-rabbit IgG secondary antibodies (Molecular Probes®, Thermo Fisher Scientific), AlexaFluor 555-conjugated donkey anti-mouse IgG secondary antibodies (Molecular Probes®, Thermo Fisher Scientific), and the DNA marker DAPI (Dojindo Laboratories, Mashiki, Kumamoto, Japan). Fluorescent images were acquired using a confocal microscope (Stellaris 5, Leica Microsystems GmbH). In this image, green fluorescence indicates staining with rabbit anti-phosphorylated p62 (SQSTM1) (Ser351) antibody, indicating activation of mitochondrial-selective autophagy (mitophagy). Red fluorescence indicates staining of ATP synthase complex V in the inner mitochondrial membrane with mouse anti-ATP5H antibody. Blue indicates staining with DAPI (nuclei), and the white line represents the scale bar. The white dotted rectangle in the low-magnification image on the left indicates the observation area in the high-magnification image on the right.

[0024] In this study, as shown in Figure 7, (A) mitochondrial ATP synthase ATP5H was detected in the hearts of wild-type mice (C57BL / 6) administered with a 4-OHT / ISR inhibitor (Control), and mitochondrial degradation by mitophagy was limited. (B) CreER T2 In the hearts of Nsp12 mice in which Nsp12 expression was induced by administering a 4-OHT / ISR inhibitor (Nsp12 induction), the detection level of the mitochondrial ATP synthase ATP5H was significantly reduced 12 days after administration, and conversely, mitophagy was activated. In other words, it was found that the induction of Nsp12 expression in mouse lungs causes mitochondrial damage in the heart. (C) CreER T2 In Nsp12 mice treated with the RdRp inhibitor EIDD-2801 (molnupiravir) together with a 4-OHT / ISR inhibitor (Nsp12 induction + EIDD-2801), the detectable level of mitochondrial ATP synthase ATP5H was restored 12 days after treatment. Therefore, it was revealed that the RdRp activity of Nsp12 can cause mitochondrial damage in the mouse heart, and that this Nsp12-induced mitochondrial damage in the mouse heart can be alleviated by the RdRp inhibitor EIDD-2801 (molnupiravir).

[0025] Example 5: Electron microscopy images showing mitochondrial damage in mouse hearts due to induction of Nsp12 expression and the therapeutic effect of EIDD-2801 (molnupiravir) 1) Transmission electron microscopy (TEM) analysis. Wild-type mice (C57 / BL / 6) and Nsp12 mice were administered the ISR inhibitor TAT-Cre gene as described in Example 4. To analyze the therapeutic effect of RdRp inhibitors on Nsp12, Nsp12 mice were administered EIDD-2801 in addition to the ISR inhibitor TAT-Cre gene. Twelve days after administration, the mouse hearts were fixed for 2 days at 4°C in a fixative containing 2% formaldehyde (Merck), 2% glutaraldehyde (Fujifilm Wako Pure Chemical), and 0.1 M cacodylate buffer (pH 7.4, Fujifilm Wako Pure Chemical), followed by preparation of 1-2 mm tissue sections for TEM analysis. The fixed tissue samples were washed four times in 0.1M cacodylate buffer for 15 minutes each and post-fixed in 2% osmium tetroxide in 0.1M cacodylate buffer. They were then washed four times in 0.1M cacodylate buffer for 15 minutes each. They were then dehydrated in ethanol (30%, 50%, 70%, 90%, and 100%) and embedded in epoxy resin (TAAB EPON 812, TAAB Laboratories Equipment Ltd., Berkshire, UK). The fixed samples were cut into 80-nm ultrathin sections using an ultrathin sectioner (UC7, Leica Microsystems GmbH) and mounted on grids (Cu 150 mesh, EM Fine Grid, Nissin EM, Tokyo, Japan). The sections were stained with 2.0% uranyl acetate in water for 10 minutes, followed by lead staining for 5 minutes at room temperature. TEM analysis was performed at room temperature using a transmission electron microscope (JEM-1400, JEOL, Tokyo, Japan) equipped with a tungsten filament at an accelerating voltage of 80 kV. TEM images were processed using Photoshop software (Photoshop 2022.23.2.1, Adobe). In this image, white arrows indicate abnormalities in the cristae structure inside mitochondria and damage caused by mitochondrial swelling and enlargement. The black line represents the scale bar.

[0026] As shown in Figure 8, TEM analysis of mouse hearts in this study revealed the following: (A) Control mice (control) treated with TAT-Cre and an ISR inhibitor from wild-type C57BL / 6 mice showed normal myocardium with clear intercalated discs and regular sarcomere structure, and numerous mitochondria. Cristae within mitochondria were also clearly observed. Therefore, mitochondrial damage caused by TAT-Cre and an ISR inhibitor administration in this study was considered to be minimal and negligible. (B) TEM analysis of hearts from Nsp12 mice treated with TAT-Cre and an ISR inhibitor to induce Nsp12 expression (Nsp12 induction) revealed significant swollen and enlarged mitochondria at 12 days after induction of Nsp12 expression. Furthermore, the cristae structure within mitochondria was disrupted, and the regularity of the sarcomere structure in the myocardium was also lost. Therefore, induction of SARS-CoV-2 Nsp12 expression in mice is considered to be a major cause of mitochondrial damage in the heart. (C) In Nsp12 mice, the RdRp activity of Nsp12 was suppressed by administering the RdRp inhibitor EIDD-2801 (molnupiravir) together with a TAT-Cre / ISR inhibitor (Nsp12 induction + EIDD-2801), and normal mitochondrial internal structure and regular myocardial sarcomere structure were observed 12 days after induction of Nsp12 expression. Therefore, the RdRp activity of Nsp12 is considered to be a major cause of mitochondrial damage in the mouse heart. This RdRp activity is therefore a promising therapeutic target for improving mitochondrial damage in the mouse heart, and we demonstrated that the RdRp inhibitor EIDD-2801 (molnupiravir) can be used to treat this condition.

[0027] Example 6: Electron microscopy images showing chronic mitochondrial damage in mouse hearts due to induction of Nsp12 expression and the therapeutic effect of EIDD-2801 (molnupiravir). Wild-type mice (C57BL / 6) and Nsp12 mice were administered the ISR inhibitor TAT-Cre as described in Example 4. Furthermore, to analyze the therapeutic effect of RdRp inhibitors on Nsp12, Nsp12 mice were administered EIDD-2801 in addition to the ISR inhibitor TAT-Cre. After 12 weeks (21 weeks for control mice), TEM analysis of chronic-phase mouse hearts was performed as described in Example 5. In this image, the white arrows indicate abnormalities in the cristae structure within mitochondria and damage due to mitochondrial swelling and enlargement. The black line represents the scale bar.

[0028] As shown in Figure 9, TEM analysis of mouse hearts revealed that (A) in wild-type (C57BL / 6) mice (control), normal myocardial sarcomere structure and intramitochondrial cristae were maintained. Therefore, the long-term effects of TAT-Cre and ISR inhibitor treatment on mitochondria in this study were considered to be minimal and negligible. (B) In Nsp12 mice, 12 weeks after Nsp12 induction with TAT-Cre and ISR inhibitor treatment, myocardial atrophy was observed, along with significant mitochondrial damage, including swelling and hypertrophy. Furthermore, intramitochondrial cristae were disrupted. Thus, early mitochondrial damage induced by Nsp12 induction persisted for a long time without recovery. Therefore, Nsp12 was identified as a cause of chronic mitochondrial damage in mouse hearts. (C) In Nsp12 mice, Nsp12 expression was induced by TAT-Cre and an ISR inhibitor, and the RdRp inhibitor EIDD-2801 (molnupiravir) was administered. Even 12 weeks after Nsp12 induction, normal myocardial sarcomere structure and mitochondrial higher-order structure were maintained, and the intramitochondrial cristae structure was also well preserved. Therefore, the RdRp activity of Nsp12 is considered to be a major cause of chronic mitochondrial damage in the mouse heart. This RdRp activity is a promising therapeutic target for improving chronic mitochondrial damage in the mouse heart. Indeed, we demonstrated that chronic mitochondrial damage can be treated with the RdRp inhibitor EIDD-2801 (molnupiravir).

[0029] According to the present invention, an RNA-dependent RNA polymerase inhibitor improves mitochondrial function that is impaired as a sequela of infectious diseases such as the novel coronavirus, and provides a method for treating symptoms caused by the impairment of said function, a pharmaceutical composition for carrying out said treatment method, and a transgenic conditional knock-in mouse designed by genetic modification to induce the expression of the RNA-dependent RNA polymerase protein Nsp12. [References] 1. Davis, HE, McCorkell, L., Vogel, JM & Topol, EJ Long COVID: major findings, mechanisms and recommendations. Nat Rev Microbiol 21, 133-146 (2023). 2. Nalbandian, A., et al. Post-acute COVID-19 syndrome. Nat Med 27, 601-615 (2021). 3. Choutka, J., Jansari, V., Hornig, M. & Iwasaki, A. Unexplained post-acute infection syndromes. Nat Med 28, 911-923 (2022). 4. Thaweethai, T., et al. Development of a definition of postacute sequelae of SARS-CoV-2 infection. JAMA 329, 1934-1946 (2023). 5. Ledford, H. Long COVID is a double curse in low-income nations - here's why. Nature 625, 20-22 (2024). 6. Mancini, DM, et al. Use of cardiopulmonary stress testing for patients with unexplained dyspnea post-coronavirus disease. JACC Heart Fail 9, 927-937 (2021). 7. Kedor, C., et al.A prospective observational study of post-COVID-19 chronic fatigue syndrome following the first pandemic wave in Germany and biomarkers associated with symptom severity. Nat Commun 13, 5104 (2022) doi: 5110.1038 / s41467-41022-32507-41466. 8. Larsen, N.W., et al. Characterization of autonomic symptom burden in long COVID: A global survey of 2,314 adults. Front Neurol 13 (2022) doi:10.3389 / fneur.2022.1012668. 9. Joseph, P., et al. Exercise pathophysiology in myalgic encephalomyelitis / chronic fatigue syndrome and postacute sequelae of SARS-CoV-2: more in common than not? Chest 164, 717-726 (2023). 10. Raj, S.R., et al. Long-COVID postural tachycardia syndrome: an American Autonomic Society statement. Clin Auton Res 31, 365-368 (2021). 11. Shouman, K., et al. Autonomic dysfunction following COVID-19 infection: an early experience. Clin Auton Res 31, 385-394 (2021). 12. Miglis, M.G., et al. A case report of postural tachycardia syndrome after COVID-19. Clin Auton Res 30, 449-451 (2020). 13.Xie, Y., Xu, E., Bowe, B. & Al-Aly, Z. Long-term cardiovascular outcomes of COVID-19. Nat Med 28, 583-590 (2022). 14. da Silva, A.L.G., et al. Impact of long COVID on the heart rate variability at rest and during deep breathing maneuver. Sci Rep 13, 22695 (2023). 15. Takahashi, T., et al. Sex differences in immune responses that underlie COVID-19 disease outcomes. Nature 588, 315-320 (2020). 16. Su, Y., et al. Multiple early factors anticipate post-acute COVID-19 sequelae. Cell 185, 881-895 e820 (2022). 17. George, P.M., et al. A persistent neutrophil-associated immune signature characterizes post-COVID-19 pulmonary sequelae. Sci Transl Med 14, eabo5795 (2022). 18. Phetsouphanh, C., et al. Immunological dysfunction persists for 8 months following initial mild-to-moderate SARS-CoV-2 infection. Nat Immunol 23, 210-216 (2022). 19. Klein, J., et al. Distinguishing features of long COVID identified through immune profiling. Nature 623, 139-148 (2023). 20. Yin, K., et al.Long COVID manifests with T cell dysregulation, inflammation and an uncoordinated adaptive immune response to SARS-CoV-2. Nat Immunol (2024) doi:10.1038 / s41590-023-01724-6 21. Peluso, M.J., et al. Multimodal molecular imaging reveals tissue-based T cell activation and viral RNA persistence for up to 2 years following COVID-19. medRxiv (2023) doi: 10.1101 / 2023.1107.1127.23293177. 22. McDonald, J.T., et al. Role of miR-2392 in driving SARS-CoV-2 infection. Cell Rep 37, 109839 (2021). 23. Guntur, V.P., et al. Signatures of mitochondrial dysfunction and impaired fatty acid metabolism in plasma of patients with Post-Acute Sequelae of COVID-19 (PASC). Metabolites 12, 1026 (2022). 24. Guarnieri, J.W., et al. Core mitochondrial genes are down-regulated during SARS-CoV-2 infection of rodent and human hosts. Sci Transl Med 15, eabq1533 (2023). 25. Appelman, B., et al. Muscle abnormalities worsen after post-exertional malaise in long COVID. Nat Commun 15, 17 (2024) doi:10.1038 / s41467-41023-44432-41463. 26. Li, S., et al. Metabolic reprogramming and epigenetic changes of vital organs in SARS-CoV-2-induced systemic toxicity. JCI Insight 6 (2021) doi: 10.1172 / jci.insight.145027. 27. Gordon, D.E., et al. A SARS-CoV-2 protein interaction map reveals targets for drug repurposing. Nature 583, 459-468 (2020). 28. Stukalov, A., et al. Multilevel proteomics reveals host perturbations by SARS-CoV-2 and SARS-CoV. Nature 594, 246-252 (2021). 29. Jiang, H.W., et al. SARS-CoV-2 proteome microarray for global profiling of COVID-19 specific IgG and IgM responses. Nat Commun 11, 3581 (2020) doi:3510.1038 / s41467-41020-17488-41468. 30. Costa-Mattioli, M. & Walter, P. The integrated stress response: From mechanism to disease. Science 368, eaa5314 (2020). 31. Malone, B., Urakova, N., Snijder, E.J. & Campbell, E.A. Structures and functions of coronavirus replication-transcription complexes and their relevance for SARS-CoV-2 drug design. Nat Rev Mol Cell Biol 23, 21-39 (2022).32. Mishina, M. & Sakimura, K. Conditional gene targeting on the pure C57BL / 6 genetic background. Neurosci Res 58, 105-112 (2007). 33. Araki, K., Araki, M., Miyazaki, J. & Vassalli, P. Site-specific recombination of a transgene in fertilized eggs by transient expression of Cre recombinase. Proc Natl Acad Sci U S A 92, 160-164 (1995).

Claims

1. A method for treating the sequelae of an RNA viral infection, comprising administering a therapeutically effective amount of an RNA-dependent RNA polymerase (RdRp) inhibitor to a subject in need thereof, including a human.

2. The method according to claim 1, wherein the sequelae are sequelae of infection with a novel coronavirus, Ebola virus, dengue virus, influenza virus, or the like.

3. The method according to claim 2, wherein the sequelae are sequelae of a novel coronavirus infection.

4. The method according to claim 3, wherein the symptoms of the sequelae are caused by organ damage due to mitochondrial dysfunction.

5. The method of claim 4, wherein the organ damage is organ damage in the lungs and / or heart.

6. The method of any one of claims 1 to 5, wherein the RdRp inhibitor is selected from the group consisting of molnupiravir, remdesivir, favipiravir, and ribavirin.

7. A pharmaceutical composition for treating the sequelae of a viral infection in a subject in need thereof, including a human, comprising a therapeutically effective amount of an RNA-dependent RNA polymerase (RdRp) inhibitor and a pharmaceutically acceptable excipient.

8. The pharmaceutical composition according to claim 7, wherein the sequelae are sequelae of infection with a novel coronavirus, Ebola virus, dengue virus, influenza virus, or the like.

9. The pharmaceutical composition according to claim 8, wherein the sequelae are sequelae of a novel coronavirus infection.

10. The pharmaceutical composition according to claim 8, wherein the symptoms of the sequelae are caused by organ damage due to mitochondrial dysfunction.

11. The pharmaceutical composition according to claim 10, wherein the organ damage is organ damage in the lungs and / or heart.

12. The pharmaceutical composition according to any one of claims 7 to 11, wherein the RdRp inhibitor is selected from the group consisting of molnupiravir, remdesivir, favipiravir, and ribavirin.

13. Transgenic conditional knock-in mice engineered by genetic modification to induce expression of the novel coronavirus RNA-dependent RNA polymerase (RdRp) protein Nsp12.

14. Lung-specific transgenic conditional knock-in mice engineered by genetic modification to induce expression of the novel coronavirus RNA-dependent RNA polymerase (RdRp) protein Nsp12.

15. A method for screening therapeutic agents for the aftereffects of novel coronavirus infection using the knock-in mouse described in claim 13.

16. A method for screening therapeutic agents for the aftereffects of novel coronavirus infection using the knock-in mouse described in claim 14.

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