Severe cardiac and skeletal manifestations in DMD-edited microminipigs: an advanced surrogate for Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is an intractable X-linked muscular dystrophy caused by mutations in the DMD gene. While many animal models have been used to study the disease, translating findings to humans has been challenging. Microminipigs, with their pronounced physiological similarity to humans and notably compact size amongst pig models, could offer a more representative model for human diseases. Here, we accomplished precise DMD modification in microminipigs by co-injecting embryos with Cas9 protein and a single-guide RNA targeting exon 23 of DMD. The DMD-edited microminipigs exhibited pronounced clinical phenotypes, including perturbed locomotion and body-wide skeletal muscle weakness and atrophy, alongside augmented serum creatine kinase levels. Muscle weakness was observed as of one month of age, respiratory and cardiac dysfunctions emerged by the sixth month, and the maximum lifespan was 29.9 months. Histopathological evaluations confirmed dystrophin deficiency and pronounced dystrophic pathology in the skeletal and myocardial tissues, demonstrating that these animals are an unprecedented model for studying human DMD. The model stands as a distinct and crucial tool in biomedical research, offering deep understanding of disease progression and enhancing therapeutic assessments, with potential to influence forthcoming treatment approaches.

Single amino acid mutation of nectin-1 provides remarkable resistance against lethal pseudorabies virus infection in mice.

An approach to genetically engineered resistance to pseudorabies virus (PRV) infection was examined by using a mouse model with defined point mutation in primary receptor for alphaherpesviruses, nectin-1, by the CRISPR/Cas9 system. It has become clear that phenylalanine at position 129 of nectin-1 is important for binding to viral glycoprotein D (gD), and mutation of phenylalanine 129 to alanine (F129A) prevents nectin-1 binding to gD and virus entry in vitro. Here, to assess the antiviral potential of the single amino acid mutation of nectin-1, F129A, in vivo, we generated genome-edited mutant mouse lines; F129A and 135 knockout (KO). The latter, 135 KO used as a nectin-1 knockout line for comparison, expresses a carboxy-terminal deleted polypeptide consisting of 135 amino acids without phenylalanine 129. In the challenge with 10 LD50 PRV via intranasal route, perfect protection of disease onset was induced by expression of the mutation of nectin-1, F129A (survival rate: 100% in F129A and 135 KO versus 0% in wild type mice). Neither viral DNA/antigens nor pathological changes were detected in F129A, suggesting that viral entry was prevented at the primary site in natural infection. In the challenge with 50 LD50 PRV, lower but still strong protective effect against disease onset was observed (survival rate: 57% in F129A and 75% in 135 KO versus 0% in wild type mice). The present results indicate that single amino acid mutation of nectin-1 F129A provides significant resistance against lethal pseudorabies.

Antiviral effects against influenza A virus infection by a short hairpin RNA targeting the non-coding terminal region of the viral nucleoprotein gene.

RNA interference (RNAi) can inhibit Influenza A virus (IAV) infection in a gene-specific manner. In this study, we constructed a transgene expressing a short hairpin RNA (shRNA) that targets the noncoding region of the IAV RNA gene encoding nucleoprotein (NP). To investigate the antiviral effects of the shRNA, we generated two transgenic mouse lines with this transgene. Unfortunately, there was no apparent difference in IAV resistance between transgenic and non-transgenic littermates. To further investigate the antiviral effects of the shRNA, we prepared mouse embryonic fibroblasts (MEFs) from transgenic and non-transgenic mice. In experimental infections using these MEFs, virus production of mouse-adapted IAV strain A/Puerto Rico/8/1934 (PR8) in the transgenic MEFs was suppressed by means of the down-regulation of the viral RNA gene transcription in the early stages of infection in comparison with non-transgenic MEFs. These results indicated that expression of the shRNA was able to confer antiviral properties against IAVs to MEFs, although the effects were limited. Our findings suggest that the shRNA targeting the noncoding region of the viral RNA (vRNA) of NP might be a supporting tool in developing influenza-resistant poultry.

Evaluation of the antiviral potential of the soluble forms of glycoprotein D receptors on ocular herpes caused by HSV-1 and HSV-2 infections in a transgenic mouse model.

Ocular herpes, caused by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) infections, remains an important corneal disease, which may result in loss of vision. Because the frequency of acyclovir resistance in HSV has increased, novel antiviral agents are needed for therapeutic approaches to ocular herpes. Several studies have demonstrated that fusion proteins containing entire ectodomain of HSV glycoprotein D receptors, including herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2, and the Fc portion of human IgG (HVEMIg, nectin-1Ig, and nectin-2Ig, respectively), can exert antiviral effects in vitro and in vivo. Here, to evaluate the antiviral potential of HVEMIg, nectin-1Ig, and nectin-2Ig against ocular infections with HSV, transgenic mice expressing these fusion proteins were ocularly inoculated with HSV-1 and HSV-2. Transgenic mouse lines expressing HVEMIg and nectin-1Ig showed marked resistance to ocular herpes; on the other hand, mouse lines expressing nectin-2Ig did not. Furthermore, to investigate the therapeutic effects of nectin-1Ig, which can neutralize HSVs in vitro against ocular disease, transgenic mouse serum containing nectin-1Ig was dropped into the eyes of wild-type mice after HSV infection. Reduction of severe symptoms could be observed in mice treated with nectin-1Ig serum. These results warrant further study of soluble HVEM and nectin-1 products as preventive and therapeutic agents against ocular herpes caused by HSV-1 and HSV-2 infections, especially nectin-1Ig as a new eye drop.

Comparison of the antiviral potential among soluble forms of herpes simplex virus type-2 glycoprotein D receptors, herpes virus entry mediator A, nectin-1 and nectin-2, in transgenic mice.

Herpesvirus entry mediator A (HVEM), nectin-1 and nectin-2 are cellular receptors of glycoprotein D (gD) of herpes simplex virus type-2 (HSV-2). It has been shown that soluble forms of HSV gD receptors have the antiviral potential in cultured cells and transgenic mice. Here, to compare antiviral potential of soluble forms of HVEM, nectin-1 and nectin-2 against HSV-2 infections in vivo, transgenic mice expressing fusion proteins consisting of the entire ectodomain of HVEM, nectin-1 or nectin-2 and the Fc portion of human IgG (HVEMIg, nectin-1Ig and nectin-2Ig, respectively) were intraperitoneally infected with HSV-2. In the infection with 3 MLD50 (50 % mouse lethal dose), effective resistance was not observed in transgenic mice expressing nectin-2Ig. In a transgenic mouse line with high expression of nectin-1Ig, significant protection from the infection with 30 and 300 MLD50 was observed (survival rate of 100 and 71 %, respectively). On the other hand, transgenic mice expressing HVEMIg showed a complete resistance to the lethal infection even with 300 MLD50 (survival rate of 100 %). These results demonstrated that HVEMIg could exert effective antiviral activities against HSV-2 infections in vivo as compared with other soluble forms of HSV gD receptors.

A soluble form of Siglec-9 provides a resistance against Group B Streptococcus (GBS) infection in transgenic mice.

Group B Streptococcus (GBS) is a leading cause of invasive bacterial infections in human newborns. A key GBS virulence factor is its capsular polysaccharide (CPS), possessing terminal sialic acid residues that suppress host immune response and provide a survival advantage to the pathogen. CPS binds to Siglec-9 expressed on neutrophils, which is expected to down-regulate the immune responsiveness of neutrophils. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of CPS to Siglec-9 on immune cells, leading to provide antibacterial benefit against GBS infection in the transgenic mouse line expressing sSiglec-9 (sSiglec-9 Tg). The sSiglec-9 in the sera of sSiglec-9 Tg bound to the sialylated-GBS strains belonging to serotypes Ia, Ib, II, III, IV and V in whole GBS cell ELISA. When GBS cells of serotype III that is a common serotype in late-onset GBS disease (LOD) were intraperitoneally inoculated into sSiglec-9 Tg, sSiglec-9 Tg showed a significant resistance as compared with non-transgenic littermates. Furthermore, GBS serotype III organisms were not detected in cultures of the blood from surviving mice (<1 × 103 CFU/ml). These results indicated that sSiglec-9 Tg mice were more efficient in eliminating GBS and survived better after the intraperitoneal challenge with GBS serotype III bacteria.

Cross-protective potential of anti-nucleoprotein human monoclonal antibodies against lethal influenza A virus infection.

The nucleoprotein (NP) possesses regions that are highly conserved among influenza A viruses, and has therefore been one of the target viral proteins for development of a universal influenza vaccine. It has been expected that human or humanized antibodies will be made available for the prophylaxis, pre-emptive and acute treatment of viral infection. However, it is still unclear whether anti-NP human antibody can confer protection against influenza virus infection. In this study, we generated transgenic mice expressing anti-NP human mAbs derived from lymphocytes of a patient infected with H5N1 highly pathogenic avian influenza (HPAI) virus, and experimental infections were conducted to examine antiviral effects of the anti-NP antibodies against H5N1 HPAI viral infections with a high fatality rate in mammals. Transgenic mouse lines expressing the anti-NP human mAbs at more than 1 mg ml-1 showed marked resistance to H5N1 virus infections. In addition, resistance to infection with an H1N1 subtype that shows strong pathogenicity to mice was also confirmed. Although the anti-NP mAbs expressed in the transgenic mice did not neutralize the virus, the mAbs could bind to NP located on the surface of infected cells. These results suggested a possibility that the non-neutralizing anti-NP human mAbs could induce indirect antiviral effects, such as antibody-dependent cellular cytotoxicity or complement-dependent cytotoxicity. Taken together, these results demonstrated that anti-NP human mAbs play an important role in heterosubtypic protection against lethal influenza virus infections in vivo.

A soluble form of human nectin-2 impairs exocrine secretion of pancreas and formation of zymogen granules in transgenic mice.

Transgenic mouse lines expressing a soluble form of human nectin-2 (hNectin-2Ig Tg) exhibited distinctive elevation of amylase and lipase levels in the sera. In this study, we aimed to clarify the histopathology and to propose the transgenic mouse lines as new animal model for characteristic pancreatic exocrine defects. The significant increase of amylase and lipase levels in sera of the transgenic lines approximately peaked at 8 weeks old and thereafter, plateaued or gradually decreased. The histopathology in transgenic acinar cells was characterized by intracytoplasmic accumulation of abnormal proteins with decrease of normal zymogen granules. The hNectin-2Ig expression was observed in the cytoplasm of pancreatic acinar cells, which was consistent with zymogen granules. However, signals of hNectin-2Ig were very weak in the transgenic acinar cells with the abnormal cytoplasmic accumulaion. The PCNA-positive cells increased in the transgenic pancreas, which suggested the affected acinar cells were regenerated. Acinar cells of hNectin-2Ig Tg had markedly small number of zymogen granules with remarkable dilation of the endoplasmic reticulum (ER) lumen containing abundant abnormal proteins. In conclusion, hNectin-2Ig Tg is proposed as a new animal model for characteristic pancreatic exocrine defects, which are due to the ER stress induced by expression of mutated cell adhesion molecule that is a soluble form of human nectin-2.

A soluble form of Siglec-9 provides an antitumor benefit against mammary tumor cells expressing MUC1 in transgenic mice.

Tumor-associated MUC1 binds to Siglec-9, which is expected to mediate tumor cell growth and negative immunomodulation. We hypothesized that a soluble form of Siglec-9 (sSiglec-9) competitively inhibits a binding of MUC1 to its receptor molecules like human Siglec-9, leading to provide antitumor benefit against MUC1-expressing tumor, and generated transgenic mouse lines expressing sSiglec-9 (sSiglec-9 Tg). When mammary tumor cells expressing MUC1 were intraperitoneally transplanted into sSiglec-9 Tg, tumor proliferation was slower with the lower histological malignancy as compared with non-transgenic mice. The sSiglec-9 was detected in the ascites caused by the tumor in the sSiglec-9 Tg, and sSiglec-9 and MUC1 were often colocalized on surfaces of the tumor cells. PCNA immunohistochemistry also revealed the reduced proliferation of the tumor cells in sSiglec-9 Tg. In sSiglec-9 Tg with remarkable suppression of tumor proliferation, MUC1 expressions were tend to be reduced. In the ascites of sSiglec-9 Tg bearing the tumor, T cells were uniformly infiltrated, whereas aggregations of degenerative T cells were often observed in the non-transgenic mice. These results suggest that sSiglec-9 has an antitumor benefit against MUC1-expressing tumor in the transgenic mice, which may avoid the negative immunomodulation and/or suppress tumor-associated MUC1 downstream signal transduction, and subsequent tumor proliferation.

Resistance to influenza A virus infection in transformed cell lines expressing an anti-PB2 monoclonal antibody.

The polymerase basic 2 (PB2) protein is one of four proteins that make up the influenza A virus replication complex, which is responsible for viral gene transcription and replication. To assess the antiviral potential of an anti-PB2 monoclonal antibody that inhibits RNA transcription of influenza A viruses, Mardin-Darby canine kidney (MDCK) cells were transformed with two transgenes that encode the light and heavy chains of the monoclonal antibody. The transformed cell lines expressing this monoclonal antibody displayed resistance to several subtypes of influenza A virus infection. In the transformed cell lines infected with influenza A virus, the level of viral RNA transcription was decreased and the effective nuclear transportation of the PB2 protein was also inhibited. These results demonstrate that the anti-PB2 intrabody is potentially able to interfere with the effective nuclear transportation of PB2 protein, resulting in the observed resistance to influenza A virus infection in vitro.

Abnormal spermatogenesis and reduced fertility in transgenic mice expressing the immediate-early protein IE180 of pseudorabies virus.

Transcription factors of alphaherpesviruses not only control the expression of their own viral genes, but also influence the gene expression of mammalian cells. In the course of breeding of the transgenic mouse line (TgIE96) expressing the immediate-early protein IE180 of pseudorabies virus belonging to the subfamily Alphaherpesvirinae, we found that TgIE96 male mice suffered from severe breeding difficulties. Testes of TgIE96 were smaller than that of non-transgenic littermates and abnormal spermatogenesis such as morphological, numerical and functional anomalies of spermatozoa were found in the transgenic mouse line. Expression of IE180 was detected in the germ cells at all stages, especially spermatocytes, and fewer Sertoli cells. In addition, expression of IE180 was also detected in the germinal cells of C57BL/6 mice inoculated with PRV into their testes. These results suggest that IE180 of PRV induces male infertility by abnormal spermatogenesis, which effect morphological, numerical, and functional anomalies of spermatozoa, in transgenic mice.

Motor-coordination-dependent learning, more than others, is impaired in transgenic mice expressing pseudorabies virus immediate-early protein IE180.

The cerebellum in transgenic mice expressing pseudorabies virus immediate-early protein IE180 (TgIE96) was substantially diminished in size, and its histoarchitecture was severely disorganized, resulting in severe ataxia. TgIE96 mice can therefore be used as an experimental model to study the involvement of cerebellar circuits in different learning tasks. The performance of three-month-old TgIE96 mice was studied in various behavioral tests, including associative learning (classical eyeblink conditioning), object recognition, spatial orientation (water maze), startle response and prepulse inhibition, and passive avoidance, and compared with that of wild-type mice. Wild-type and TgIE96 mice presented similar reflexively evoked eyeblinks, and acquired classical conditioned eyelid responses with similar learning curves for both trace and delay conditioning paradigms. The two groups of mice also had similar performances during the object recognition test. However, they showed significant differences for the other three tests included in this study. Although both groups of animals were capable of swimming, TgIE96 mice failed to learn the water maze task during the allowed time. The startle response to a severe tone was similar in both control and TgIE96 mice, but the latter were unable to produce a significant prepulse inhibition. TgIE96 mice also presented evident deficits for the proper accomplishment of a passive avoidance test. These results suggest that the cerebellum is not indispensable for the performance of classical eyeblink conditioning and for object recognition tasks, but seems to be necessary for the proper performance of water maze, prepulse inhibition, and passive avoidance tests.