Elevated expression of miR-301a and its functional roles in canine oral melanoma.

miR-301a is one of numerous dysregulated microRNAs (miRNAs) in canine oral melanoma (COM), one of which is miR-301a (upregulated). Its biological role has been described in various human cancer types, including malignant melanoma, but not in COM. Accordingly, in this study, we investigated miR-301a expression in COM in greater detail to ascertain whether it could serve as a diagnostic biomarker, elucidate its functional roles in this cancer, and predict the possible pathways by which it exerts its effects. Relative expression of miR-301a was investigated in clinical oral tissue and plasma samples and COM cell (KMeC and LMeC) lines using qRT-PCR. Knockdown of miR-301a was also validated for KMeC and LMeC cells using qRT-PCR. We performed CCK-8 assays to assess cell proliferation, monolayer wound-healing, and transwell migration assays to assess cell migration, a colony-formation assay to assess clonogenicity, a TUNEL assay and flow cytometry to assess apoptosis-related effects, and gene enrichment analyses to predict possible related pathways. miR-301a was markedly upregulated in COM oral tissue and plasma clinically, suggesting its potential as a diagnostic biomarker for COM diagnosis. In vitro assays demonstrated that miR-301 significantly inhibited apoptosis in COM cells while promoting cell migration, proliferation, and clonogenicity. We also predicted that miR-301 exerts cancer-promoting effects through the Wnt signalling pathway for COM. Our findings suggest that miR-301a is a COM oncomiR that regulates several oncogenic phenotypes with the potential to be a diagnostic biomarker.

Effects of neddylation on viral infection: an overview.

Neddylation is a post-translational modification that plays an important role not only in cancer development but also in regulating viral infection and replication. Upregulation of neddylation occurs in viral infections, and inhibition of neddylation can suppress viral replication. Neddylation is thought to enhance viral protein stability and replication. Neddylation has been reported to enhance the stability of the regulatory hepatitis B virus (HBV) X protein, modulate viral replication, and enhance hepatocarcinogenesis. Inhibition of neddylation using the NEDD8-activating enzyme E1 inhibitor MLN4924 inhibits viral replication, including that of HBV. Understanding of the role of neddylation in viral infections is critical for developing new therapeutic targets and potential treatment strategies. In this review, we discuss recent progress in the understanding of the effects of neddylation during viral infection, particularly in HBV infection, and strategies for curing viral infection by targeting the neddylation pathway.

Investigation of koala retrovirus in captive koalas with pneumonia and comparative analysis of subtype distribution.

This study focused on the involvement of koala retrovirus (KoRV) in pneumonia in koalas. Three deceased pneumonic koalas from a Japanese zoo were examined in this study. Hematological and histopathological findings were assessed, and KoRV proviral DNA loads in the blood and tissues were compared with those of eight other KoRV-infected koalas from different zoos. Demographic data and routine blood profiles were collected, and blood and tissue samples were analyzed to rule out concurrent infections in pneumonic koalas. KoRV subtyping and measurement of the KoRV proviral DNA load were performed by polymerase chain reaction (PCR) using specific primers targeting the pol and env genes. The results showed that the koalas had histopathologically suppurative and fibrinous pneumonia. Chlamydiosis was not detected in any of the animals. PCR analysis revealed KoRV-A, -B, and -C infections in all koalas, except for animals K10-11, which lacked KoRV-B. Significant variations in the proviral DNA loads of these KoRV subtypes were observed in all tissues and disease groups. Most tissues showed reduced KoRV loads in koalas with pneumonia, except in the spleen, which had significantly higher loads of total KoRV (2.54 × 107/µg DNA) and KoRV-A (4.74 × 107/µg DNA), suggesting potential immunosuppression. This study revealed the intricate dynamics of KoRV in various tissues, indicating its potential role in koala pneumonia via immunosuppression and opportunistic infections. Analysis of the levels of KoRV proviral DNA in different tissues will shed light on viral replication and the resulting pathogenesis in future studies.

Molecular insights of a CBP/ß-catenin-signaling inhibitor on nonalcoholic steatohepatitis-induced liver fibrosis and disorder.

Nonalcoholic steatohepatitis (NASH) is a progressive fibrotic disease associated with an increased risk of developing hepatocellular carcinoma; at present, no efficient therapeutic strategy has been established. Herein, we examined the efficacy of PRI-724, a potent inhibitor of CBP/ß-catenin signaling, for treating NASH-related liver fibrosis and disorder and characterized its mechanism. Choline-deficient, L-amino acid-defined, high-fat diet (CDAHFD)-fed mice exhibited NASH-induced liver fibrosis that is characterized by steatosis, lobular inflammation, hepatocellular injury and collagen fibrils. To examine the therapeutic effect, CDAHFD-fed mice were administered PRI-724. Serum levels of ALT and pro-fibrotic molecule, i.e. Mac-2 bp, alpha smooth muscle actin, type I and type III collagens, decreased significantly. mRNA levels of the matrix metalloproteinases Mmp8 and Mmp9 in the liver were significantly increased, and increases in the abundance of MMP9-producing neutrophils and macrophages were observed. Marco+Mmp9+Cd68+ Kupffer cells were only observed in the livers of mice treated with PRI-724, and Mmp9 expression in Marco+Cd68+ Kupffer cells increased 4.3-fold. Moreover, hepatic expression of the lipid metabolism regulator, pyruvate dehydrogenase kinase 4 and liver lipid droplets also decreased significantly. PRI-724-treated NASH mice not only recovered from NASH-related liver fibrosis through the effect of PRI-724 down-regulating the expression of pro-fibrotic genes and up-regulating the expression of anti-fibrotic genes, but they also recovered from NASH-induced liver disorder. PRI-724, a selective CBP/ß-catenin inhibitor, thus shows a potent therapeutic effect for NASH-related liver fibrosis and for decreasing adipose tissue in the liver.

Newly identified cytochrome P450 3A genes of tree shrews and pigs are expressed and encode functional enzymes.

Novel cytochrome P450 3A5 (CYP3A5) cDNA in tree shrews (which are non-rodent primate-like species) and pig CYP3A227 cDNA were identified, along with known pig CYP3A22, CYP3A29, and CYP3A46 cDNAs. All five cDNAs contained open reading frames encoding a polypeptide of 503 amino acids that shared high sequence identity (72-78 %) with human CYP3A4 and were more closely related to human CYP3As than rat CYP3As by phylogenetic analysis. CYP3A5 was the only CYP3A in the tree shrew genome, but pig CYP3A genes formed a CYP3A gene cluster in the genomic region corresponding to that of human CYP3A genes. Tree shrew CYP3A5 mRNA was predominantly expressed in liver and small intestine, among the tissues analyzed, whereas pig CYP3A227 mRNA was most abundantly expressed in jejunum, followed by liver. Metabolic assays established that tree shrew CYP3A5 and pig CYP3A proteins heterologously expressed in Escherichia coli metabolized typical human CYP3A4 substrates nifedipine and midazolam. These results suggest that novel tree shrew CYP3A5 and pig CYP3A227 were functional enzymes able to metabolize human CYP3A4 substrates in liver and small intestine, similar to human CYP3A4, although pig CYP3A227 mRNA was minimally expressed in all tissues analyzed.

Regulatory Role of Ribonucleotide Reductase Subunit M2 in Hepatocyte Growth and Pathogenesis of Hepatitis C Virus.

Hepatitis C virus (HCV) frequently causes chronic infection in the human liver, which may progress to advanced hepatic fibrosis, cirrhosis, and hepatocellular carcinoma. HCV primarily infects highly differentiated quiescent hepatocytes and can modulate cell cycle-regulatory genes and proliferation pathways, which ultimately contribute to persistent infection and pathogenesis. On the other hand, several studies have shown differential regulation of HCV RNA and viral protein expression levels, depending on the proliferation state of hepatocytes and the phase of the cell cycle. HCV typically requires factors provided by host cells for efficient and persistent viral replication. Previously, we found that HCV infection upregulates the expression of ribonucleotide reductase subunit M2 (RRM2) in quiescent hepatocytes. RRM2 is a rate-limiting protein that catalyzes de novo synthesis of deoxyribonucleotide triphosphates, and its expression is highly regulated during various phases of the cell cycle. RRM2 functions as a pro-viral factor essential for HCV RNA synthesis, but its functional role in HCV-induced liver diseases remains unknown. Here, we present a comprehensive review of the role of the hepatocyte cell cycle, in correlation with RRM2 expression, in the regulation of HCV replication. We also discuss the potential relevance of this protein in the pathogenesis of HCV, particularly in the development of hepatocellular carcinoma.

Newly Identified Tree Shrew Cytochrome P450 2A13 is Expressed in Liver and Lung and Encodes a Functional Drug-Metabolizing Enzyme Similar to Dog Cytochrome P450 2A13 and Pig Cytochrome P450 2A19.

The tree shrew, a non-rodent primate-like species, is used in various fields of biomedical research, including hepatitis virus infection, myopia, depression, and toxicology. Recent genome analysis found that the numbers of cytochrome P450 (P450 or CYP) genes are similar in tree shrews and humans and their sequence identities are high. Although the P450s are a family of important drug-metabolizing enzymes, they have not yet been fully investigated in tree shrews. In the current study, tree shrew CYP2A13 cDNA was isolated from liver, and its characteristics were compared with those of pig, dog, and human CYP2As. Tree shrew CYP2A13 amino acid sequences were highly identical (87-92%) to the human CYP2As and contained sequence motifs characteristic of P450s. Phylogenetic analysis revealed that tree shrew CYP2A13 was more closely related to human CYP2As than to rat CYP2As, similar to dog and pig CYP2As. Among the tissue types analyzed, tree shrew CYP2A13 mRNA was preferentially expressed in liver and lung, similar to dog CYP2A13 mRNA, whereas dog CYP2A25 and pig CYP2A19 mRNAs were predominantly expressed in liver. Tree shrew liver microsomes and tree shrew CYP2A13 proteins heterologously expressed in Escherichia coli catalyzed coumarin 7-hydroxylation and phenacetin O-deethylation, just as human, dog, and pig CYP2A proteins and liver microsomes do. These results demonstrate that tree shrew CYP2A13 is expressed in liver and lung and encodes a functional drug-metabolizing enzyme. SIGNIFICANCE STATEMENT: Novel tree shrew cytochrome P450 2A13 (CYP2A13) was identified and characterized in comparison with human, dog, and pig CYP2As. Tree shrew CYP2A13 isolated from liver had high sequence identities and close phylogenetic relationships to its human homologs and was abundantly expressed in liver and lung at the mRNA level. Tree shrew CYP2A13 metabolized coumarin and phenacetin, human selective CYP2A6 and CYP2A13 substrates, respectively, similar to dog and pig CYP2As, and is a functional drug-metabolizing enzyme likely responsible for drug clearances.

Novel cytochrome P450 1 (CYP1) genes in tree shrews are expressed and encode functional drug-metabolizing enzymes.

Tree shrews (Tupaia belangeri) are a non-rodent primate-like species sometimes used for biomedical research involving hepatitis virus infections and toxicology. Genome analysis has indicated similarities between tree shrews and humans in the numbers of cytochromes P450 (P450 or CYP), which constitute a family of important drug-metabolizing enzymes; however, P450s have not been fully investigated in tree shrews. In this study, we identified CYP1A1, CYP1A2, CYP1B1, and CYP1D1 cDNAs from tree shrew liver and compared their characteristics with dog, pig, and human CYP1As. The deduced amino acid sequences of tree shrew CYP1s were highly identical (82-87 %) to human CYP1s. In tree shrews, CYP1A1 and CYP1A2 mRNAs were preferentially expressed in liver, whereas CYP1D1 mRNA was preferentially expressed in kidney and lung. In contrast, CYP1B1 mRNA was expressed in various tissues, with the most abundant expression in spleen. Among the tree shrew CYP1 mRNAs, CYP1A2 mRNA was most abundant in liver, and CYP1B1 mRNA was most abundant in kidney, small intestine, and lung. All tree shrew CYP1 proteins heterologously expressed in Escherichia coli catalyzed caffeine and estradiol in a similar manner to tree shrew liver microsomes and human, dog, and pig CYP1 proteins. These results suggest that tree shrew CYP1A1, CYP1A2, CYP1B1, and CYP1D1 genes, different form human pseudogene CYP1D1P, are expressed in liver, small intestine, lung, and/or kidney and encode functional drug-metabolizing enzymes important in toxicology.

In vivo Delivery Tools for Clustered Regularly Interspaced Short Palindromic Repeat/Associated Protein 9-Mediated Inhibition of Hepatitis B Virus Infection: An Update.

Chronic hepatitis B virus (HBV) infection remains a major global health problem despite the availability of an effective prophylactic HBV vaccine. Current antiviral therapies are unable to fully cure chronic hepatitis B (CHB) because of the persistent nature of covalently closed circular DNA (cccDNA), a replicative template for HBV, which necessitates the development of alternative therapeutic approaches. The CRISPR/Cas system, a newly emerging genome editing tool, holds great promise for genome editing and gene therapy. Several in vitro and/or in vivo studies have demonstrated the effectiveness of HBV-specific clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (CRISPR/Cas9) systems in cleaving HBV DNA and cccDNA. Although recent advances in CRISPR/Cas technology enhance its prospects for clinical application against HBV infection, in vivo delivery of the CRISPR/Cas9 system at targets sites remains a major challenge that needs to be resolved before its clinical application in gene therapy for CHB. In the present review, we discuss CRISPR/Cas9 delivery tools for targeting HBV infection, with a focus on the development of adeno-associated virus vectors and lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery to treat CHB. In addition, we discuss the importance of delivery tools in the enhancement of the antiviral efficacy of CRISPR/Cas9 against HBV infection.

Subtype distribution and expression of the koala retrovirus in the Japanese zoo koala population.

We investigated the proviral copies and RNA expression in koala retrovirus (KoRV)-infected koalas. To ascertain any variation in viral load by institution, age, sex, or body condition score, we quantified KoRV proviral DNA and RNA loads in captive koalas (n = 37) reared in Japanese zoos. All koalas were positive for KoRV genes (pol, LTRs, and env of KoRV-A) in genomic DNA (gDNA), and 91.89% were positive for the pol gene in RNA. In contrast, the distribution rates of KoRV-B, KoRV-C, KoRV-D, and KoRV-F env genes in gDNA were 94.59%, 27.03%, 67.57%, and 54.05%, respectively. A wide inter-individual variation and/or a significant inter-institutional difference in proviral DNA (p < 0.0001) and RNA (p < 0.001) amounts (copies/103 koala ß-actin copies) were observed in Awaji Farm England Hill Zoo koalas, which were obtained from southern koala populations, suggesting exogenous incorporation of KoRV in these koalas. Significant (p < 0.05) age differences were noted in KoRV RNA load (p < 0.05) and median total RNA load (p < 0.001), with loads higher in younger koalas (joeys and juveniles). Thus, the current study provides the distribution of KoRV subtypes in Japanese zoo koala populations and identifies several additional risk factors (sex, age, and body condition) associated with KoRV expression.

Toll-like Receptor Response to Hepatitis C Virus Infection: A Recent Overview.

Hepatitis C virus (HCV) infection remains a major global health burden, causing chronic hepatitis, cirrhosis, and hepatocellular carcinoma. Toll-like receptors (TLRs) are evolutionarily conserved pattern recognition receptors that detect pathogen-associated molecular patterns and activate downstream signaling to induce proinflammatory cytokine and chemokine production. An increasing number of studies have suggested the importance of TLR responses in the outcome of HCV infection. However, the exact role of innate immune responses, including TLR response, in controlling chronic HCV infection remains to be established. A proper understanding of the TLR response in HCV infection is essential for devising new therapeutic approaches against HCV infection. In this review, we discuss the progress made in our understanding of the host innate immune response to HCV infection, with a particular focus on the TLR response. In addition, we discuss the mechanisms adopted by HCV to avoid immune surveillance mediated by TLRs.

Blocking neddylation elicits antiviral effect against hepatitis B virus replication.

BACKGROUND: Hepatitis B Virus (HBV) is the most common cause of chronic liver disease worldwide. The mechanisms that regulate HBV viral replication remain poorly defined. Here, we show that blocking of the neddylation elicits antiviral effect against HBV replication, indicating that NEDD8 supports viral production. METHODS AND RESULTS: To explore role of neddylation, HBV-replicating HepG2.2.15.7 cells and HBV-infected HepG2-hNTCP-30 cells were treated with siNEDD8 and MLN4924, a potent and selective NEDD8-activating enzyme inhibitor. Cell viability, intracellular and extracellular HBV DNA, covalently closed circular DNA (cccDNA), HBsAg, HBeAg, and HBcrAg were measured to assess the consequences of the various treatments on viral replication. Our data showed that HBV infection increased NEDD8 expression in human liver cell lines. Symmetrically, NEDD8 knockdown by siRNA or MLN4924 treatments decreased HBV replication in HepG2.2.15.7 and HepG2-hNTCP-30 cells. Notably, HBsAg, and HBeAg secretions were strongly suppressed in the culture supernatants, but not the HBcrAg. These results indicate that the suppression of NEDD8 decreases HBV replication. However, cccDNA steady level confirms once again its persistence and longevity in chronic infection. CONCLUSION: The manipulation of the neddylation pathway can thus provide new tools interfering with HBV persistence as well as novel therapeutic strategies against chronic hepatitis B.

Longer Poly(U) Stretches in the 3'UTR Are Essential for Replication of the Hepatitis C Virus Genotype 4a Clone in in vitro and in vivo.

The 3' untranslated region (UTR) of the hepatitis C virus (HCV) genome plays a significant role in replication including the poly(U) tract (You and Rice, 2008). Here we established an HCV clone that is infectious in vitro and in vivo, from an Egyptian patient with chronic HCV infection and hepatocellular carcinoma (HCC). First, we inoculated the patient plasma into a humanized chimeric mouse and passaged. We observed HCV genotype 4a propagation in the chimeric mouse sera at 1.7 × 107 copies/mL after 6 weeks. Next, we cloned the entire HCV sequence from the HCV-infected chimeric mouse sera using RT-PCR, and 5' and 3' RACE methodologies. We obtained first a shorter clone (HCV-G4 KM short, GenBank: AB795432.1), which contained 9,545 nucleotides with 341 nucleotides of the 5'UTR and 177 nucleotides of the 3'UTR, and this was frequently obtained for unknown reasons. We also obtained a longer clone by dividing the HCV genome into three fragments and the poly (U) sequences. We obtained a longer 3'UTR sequence than that of the HCV-G4 KM short clone, which contained 9,617 nucleotides. This longer clone possessed a 3'-UTR of 249 nucleotides (HCV-G4 KM long, GenBank: AB795432.2), because of a 71-nucleotide longer poly (U) stretch. The HCV-G4-KM long clone, but not the HCV-G4-KM short clone, could establish infection in human hepatoma HuH-7 cells. HCV RNAs carrying a nanoluciferase (NL) reporter were also constructed and higher replication activity was observed with G4-KM long-NL in vitro. Next, both short and long RNAs were intra-hepatically injected into humanized chimeric mice. Viral propagation was only observed for the chimeric mouse injected with the HCV-G4 KM long RNA in the sera after 21 days (1.64 × 106 copies/mL) and continued until 10 weeks post inoculation (wpi; 1.45-4.74 × 107 copies/mL). Moreover, sequencing of the HCV genome in mouse sera at 6 wpi revealed the sequence of the HCV-G4-KM long clone. Thus, the in vitro and in vivo results of this study indicate that the sequence of the HCV-G4-KM long RNA is that of an infectious clone.

Tree Shrew as an Emerging Small Animal Model for Human Viral Infection: A Recent Overview.

Viral infection is a global public health threat causing millions of deaths. A suitable small animal model is essential for viral pathogenesis and host response studies that could be used in antiviral and vaccine development. The tree shrew (Tupaia belangeri or Tupaia belangeri chinenesis), a squirrel-like non-primate small mammal in the Tupaiidae family, has been reported to be susceptible to important human viral pathogens, including hepatitis viruses (e.g., HBV, HCV), respiratory viruses (influenza viruses, SARS-CoV-2, human adenovirus B), arboviruses (Zika virus and dengue virus), and other viruses (e.g., herpes simplex virus, etc.). The pathogenesis of these viruses is not fully understood due to the lack of an economically feasible suitable small animal model mimicking natural infection of human diseases. The tree shrew model significantly contributes towards a better understanding of the infection and pathogenesis of these important human pathogens, highlighting its potential to be used as a viable viral infection model of human viruses. Therefore, in this review, we summarize updates regarding human viral infection in the tree shrew model, which highlights the potential of the tree shrew to be utilized for human viral infection and pathogenesis studies.

Macrocyclic peptides exhibit antiviral effects against influenza virus HA and prevent pneumonia in animal models.

Most anti-influenza drugs currently used, such as oseltamivir and zanamivir, inhibit the enzymatic activity of neuraminidase. However, neuraminidase inhibitor-resistant viruses have already been identified from various influenza virus isolates. Here, we report the development of a class of macrocyclic peptides that bind the influenza viral envelope protein hemagglutinin, named iHA. Of 28 iHAs examined, iHA-24 and iHA-100 have inhibitory effects on the in vitro replication of a wide range of Group 1 influenza viruses. In particular, iHA-100 bifunctionally inhibits hemagglutinin-mediated adsorption and membrane fusion through binding to the stalk domain of hemagglutinin. Moreover, iHA-100 shows powerful efficacy in inhibiting the growth of highly pathogenic influenza viruses and preventing severe pneumonia at later stages of infection in mouse and non-human primate cynomolgus macaque models. This study shows the potential for developing cyclic peptides that can be produced more efficiently than antibodies and have multiple functions as next-generation, mid-sized biomolecules.

Toll-Like Receptor and Cytokine Responses to Infection with Endogenous and Exogenous Koala Retrovirus, and Vaccination as a Control Strategy.

Koala populations are currently declining and under threat from koala retrovirus (KoRV) infection both in the wild and in captivity. KoRV is assumed to cause immunosuppression and neoplastic diseases, favoring chlamydiosis in koalas. Currently, 10 KoRV subtypes have been identified, including an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). The host's immune response acts as a safeguard against pathogens. Therefore, a proper understanding of the immune response mechanisms against infection is of great importance for the host's survival, as well as for the development of therapeutic and prophylactic interventions. A vaccine is an important protective as well as being a therapeutic tool against infectious disease, and several studies have shown promise for the development of an effective vaccine against KoRV. Moreover, CRISPR/Cas9-based genome editing has opened a new window for gene therapy, and it appears to be a potential therapeutic tool in many viral infections, which could also be investigated for the treatment of KoRV infection. Here, we discuss the recent advances made in the understanding of the immune response in KoRV infection, as well as the progress towards vaccine development against KoRV infection in koalas.

Koala retrovirus (KoRV) subtypes and their impact on captive koala (Phascolarctos cinereus) health.

Koala retrovirus (KoRV), a major pathogen of koalas, exists in both endogenous (KoRV-A) and exogenous forms (KoRV-B to J). However, the impact of infection with multiple subtypes is not well understood. Accordingly, in this study, we surveyed a representative sample from a Japanese zoo population to determine the infection status for three KoRV subtypes (KoRV-A, B, and C) and to investigate the proviral and RNA load profiles in animals with single- and multiple-subtype infections, using peripheral blood mononuclear cells (PBMCs) and plasma. Six koalas were evaluated in the study; all were infected with KoRV-A, and two koalas were coinfected with non-A subtypes (KoRV-B and/or KoRV-C). The highest KoRV total RNA and viral loads in PBMCs and plasma were found in a koala infected with multiple subtypes (KoRV-A, -B and -C). The other koala infected with multiple subtypes (KoRV-A and B) showed the highest proviral PBMC load but the lowest RNA copy number in PBMC and plasma. PBMCs from this animal were cultured for further investigation, and KoRV RNA was detected in the cells and culture supernatant after 7 and/or 14 days. The koalas harboring multiple subtypes had a higher white blood cell count than those harboring only KoRV-A and were judged to be leukemic, and they subsequently died due to lymphoma. Accordingly, we conclude that coinfection with multiple KoRV subtypes may be linked to more-severe disease. In a sequence alignment, the detected KoRV-A env gene showed 100% sequence identity to the reference gene, whereas the KoRV-B and -C env genes varied from their reference sequences.

Lipid nanoparticles loaded with ribonucleoprotein-oligonucleotide complexes synthesized using a microfluidic device exhibit robust genome editing and hepatitis B virus inhibition.

The clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) system has considerable therapeutic potential for use in treating a wide range of intractable genetic and infectious diseases including hepatitis B virus (HBV) infections. While non-viral delivery technologies for the CRISPR/Cas system are expected to have clinical applications, difficulties associated with the clinically relevant synthesis of formulations and the poor efficiency of delivery severely hinder therapeutic genome editing. We report herein on the production of a lipid nanoparticle (LNP)-based CRISPR/Cas ribonucleoprotein (RNP) delivery nanoplatform synthesized using a clinically relevant mixer-equipped microfluidic device. DNA cleavage activity and the aggregation of Cas enzymes was completely avoided under the optimized synthetic conditions. The optimized formulation, which was identified through 2 steps of design of experiments, exhibited excellent gene disruption (up to 97%) and base substitution (up to 23%) without any apparent cytotoxicity. The addition of negative charges to the RNPs by complexing single-stranded oligonucleotide (ssON) significantly enhanced the delivery of both Cas9 and Cpf1 RNPs. The optimized formulation significantly suppressed both HBV DNA and covalently closed circular DNA (cccDNA) in HBV-infected human liver cells compared to adeno-associated virus type 2 (AAV2). These findings represent a significant contribution to the development of CRISPR/Cas RNP delivery technology and its practical applications in genome editing therapy.

CD4, CD8b, and Cytokines Expression Profiles in Peripheral Blood Mononuclear Cells Infected with Different Subtypes of KoRV from Koalas (Phascolarctos cinereus) in a Japanese Zoo.

Koala retrovirus (KoRV) poses a major threat to koala health and conservation, and currently has 10 identified subtypes: an endogenous subtype (KoRV-A) and nine exogenous subtypes (KoRV-B to KoRV-J). However, subtype-related variations in koala immune response to KoRV are uncharacterized. In this study, we investigated KoRV-related immunophenotypic changes in a captive koala population (Hirakawa zoo, Japan) with a range of subtype infection profiles (KoRV-A only vs. KoRV-A with KoRV-B and/or -C), based on qPCR measurements of CD4, CD8b, IL-6, IL-10 and IL-17A mRNA expression in unstimulated and concanavalin (Con)-A-stimulated peripheral blood mononuclear cells (PBMCs). Although CD4, CD8b, and IL-17A expression did not differ between KoRV subtype infection profiles, IL-6 expression was higher in koalas with exogenous infections (both KoRV-B and KoRV-C) than those with the endogenous subtype only. IL-10 expression did not significantly differ between subtype infection profiles but did show a marked increase-accompanying decreased CD4:CD8b ratio-in a koala with lymphoma and co-infected with KoRV-A and -B, thus suggesting immunosuppression. Taken together, the findings of this study provide insights into koala immune response to multiple KoRV subtypes, which can be exploited for the development of prophylactic and therapeutic interventions for this iconic marsupial species.

Development of an in vivo delivery system for CRISPR/Cas9-mediated targeting of hepatitis B virus cccDNA.

Chronic hepatitis B virus (HBV) infection constitutes a global health issue with limited current therapeutic efficacy owing to the persistence of viral episomal DNA (cccDNA). The CRISPR/Cas9 system, a newly developed, powerful tool for genome editing and potential gene therapy, requires efficient delivery of CRISPR components for successful therapeutic application. Here, we investigated the effects of lentiviral- or adeno-associated virus 2 (AAV2) vector-mediated delivery of 3 guide (g)RNAs/Cas9 selected from 16 gRNAs. These significantly suppressed HBV replication in cells, with WJ11/Cas9 exhibiting highest efficacy and chosen for in vivo study. AAV2/WJ11-Cas9 also significantly inhibited HBV replication and significantly reduced cccDNA in the tested cells. Moreover, AAV2/WJ11-Cas9 enhanced entecavir effects when used in combination, indicative of different modes of action. Notably, in humanized chimeric mice, AAV2/WJ11-Cas9 significantly suppressed HBcAg, HBsAg, and HBV DNA along with cccDNA in the liver tissues without significant cytotoxicity; accordingly, next generation sequencing data showed no significant genomic mutations. To our knowledge, this represents the first evaluation of the CRISPR/Cas9 system using an HBV natural infection mode. Therefore, WJ11/Cas9 delivered by comparatively safer AAV2 vectors may provide a new therapeutic strategy for eliminating HBV infection and serve as an effective platform for curing chronic HBV infection.