Comprehensive Evaluation of Lipid Nanoparticles and Polyplex Nanomicelles for Muscle-Targeted mRNA Delivery.

The growing significance of messenger RNA (mRNA) therapeutics in diverse medical applications, such as cancer, infectious diseases, and genetic disorders, highlighted the need for efficient and safe delivery systems. Lipid nanoparticles (LNPs) have shown great promise for mRNA delivery, but challenges such as toxicity and immunogenicity still remain to be addressed. In this study, we aimed to compare the performance of polyplex nanomicelles, our original cationic polymer-based carrier, and LNPs in various aspects, including delivery efficiency, organ toxicity, muscle damage, immune reaction, and pain. Our results showed that nanomicelles (PEG-PAsp(DET)) and LNPs (SM-102) exhibited distinct characteristics, with the former demonstrating relatively sustained protein production and reduced inflammation, making them suitable for therapeutic purposes. On the other hand, LNPs displayed desirable properties for vaccines, such as rapid mRNA expression and potent immune response. Taken together, these results suggest the different potentials of nanomicelles and LNPs, supporting further optimization of mRNA delivery systems tailored for specific purposes.

Anti-Inflammatory Therapy for Temporomandibular Joint Osteoarthritis Using mRNA Medicine Encoding Interleukin-1 Receptor Antagonist.

Messenger RNA (mRNA) is an emerging drug modality for protein replacement therapy. As mRNA efficiently provides protein expression in post-mitotic cells without the risk of insertional mutagenesis, direct delivery of mRNA can be applied, not only as an alternative to gene therapy, but also for various common diseases such as osteoarthritis (OA). In this study, using an mRNA-encoding interleukin-1 receptor antagonist (IL-1Ra), we attempted anti-inflammatory therapy in a rat model of the temporomandibular joint (TMJ) OA, which causes long-lasting joint pain with chronic inflammation. For the intra-articular injection of mRNA, a polyplex nanomicelle, our original polymer-based carrier, was used to offer the advantage of excellent tissue penetration with few immunogenic responses. While the protein expression was transient, a single administration of IL-1Ra mRNA provided sustained pain relief and an inhibitory effect on OA progression for 4 weeks. The mRNA-loaded nanomicelles provided the encoded protein diffusely in the disc and articular cartilage without upregulation of the expression levels of the pro-inflammatory cytokines IL-6 and tumor necrosis factor-α (TNF-α). This proof-of-concept study demonstrates how anti-inflammatory proteins delivered by mRNA delivery using a polyplex nanomicelle could act to alleviate OA, stimulating the development of mRNA therapeutics.

Possible Role of Cytochrome P450 1B1 in the Mechanism of Gemcitabine Resistance in Pancreatic Cancer.

Patient-derived xenograft models reportedly represent original tumor morphology and gene mutation profiles. In addition, patient-derived xenografts are expected to recapitulate the parental tumor drug responses. In this study, we analyzed the pathways involved in gemcitabine resistance using patient-derived xenograft models of pancreatic cancer. The patient-derived xenograft models were established using samples from patients with pancreatic cancer. The models were treated with gemcitabine to better understand the mechanism of resistance to this anti-cancer drug. We performed comparative gene analysis through the next-generation sequencing of tumor tissues from gemcitabine-treated or non-treated patient-derived xenograft mice and gene set enrichment analysis to analyze mRNA profiling data. Pathway analysis of gemcitabine-treated patient-derived xenografts disclosed the upregulation of multiple gene sets and identified several specific gene pathways that could potentially be related to gemcitabine resistance in pancreatic cancer. Further, we conducted an in vitro analysis to validate these results. The mRNA expression of cytochrome P450 1B1 and cytochrome P450 2A6 was upregulated in a concentration-dependent manner following gemcitabine treatment. Moreover, the sensitivity to gemcitabine increased, and viable cells were decreased by the cytochrome P450 1B1 inhibitor, indicating that the cytochrome P450 1B1 pathway may be related to gemcitabine resistance in pancreatic cancer.

High levels of human epididymis protein 4 mRNA and protein expression are associated with chemoresistance and a poor prognosis in pancreatic cancer.

Pancreatic cancer is associated with an exceedingly poor prognosis, warranting the development of novel therapeutic strategies and discovery of prognostic predictors. Given that chemoresistance­related molecules are reportedly associated with the poor prognosis of pancreatic cancer, the present study aimed to identify molecules that could be efficacious therapeutic targets for pancreatic cancer. First, 10 patient­derived xenografts (PDXs) were established from patients with pancreatic cancer. Subsequently, after treating tumor tissue generated from the PDXs with standard drugs, next­generation sequencing (NGS) was performed using these tissues. The results of NGS analysis and immunohistochemical analysis on 80 pancreatic cancer tissues revealed that human epididymis protein 4 (HE4) expression in the anticancer drug­treated PDX group was higher than that in the untreated PDXs. In addition, chemoresistance ability was observed in tumor cell lines overexpressing HE4. Furthermore, Kaplan­Meier analysis of tumor tissues from 80 patients with pancreatic cancer was performed and it was found that patients with a high HE4 expression level had a poor survival rate compared with those who had a low HE4 expression level. Multivariate analysis also indicated the high expression level of HE4 was an independent poor prognostic biomarker. Thus, it was concluded that high gene and protein expression levels of HE4 mediate chemoresistance and are independent prognostic factors for pancreatic cancer.

High expression levels of polymeric immunoglobulin receptor are correlated with chemoresistance and poor prognosis in pancreatic cancer.

Pancreatic cancer has extremely poor prognosis, warranting the discovery of novel therapeutic and prognostic markers. The expression of polymeric immunoglobulin receptor (pIgR), a key component of the mucosal immune system, is increased in several cancers. However, its clinical relevance in pancreatic cancer remains unclear. In the present study, the prognostic value of pIgR in pancreatic cancer patients after surgical resection was assessed and it was determined that the expression of pIgR was correlated with poor prognosis. Ten pancreatic cancer patient­derived xenograft (PDX) lines were established, followed by next­generation sequencing of tumor tissues from these lines after standard chemotherapy. Immunohistochemical analysis of chemoresistance­related molecules using 77 pancreatic cancer tissues was also performed. The expression of pIgR mRNA in the PDX group treated with anticancer drugs was higher than in the untreated group. High pIgR expression in tissue specimens from 77 pancreatic cancer patients was significantly associated with poor prognosis and was revealed to be an independent prognostic factor, predicting poor outcomes. High pIgR mRNA and protein levels were independent prognostic factors, indicating that pIgR could be a novel predictor for poor prognosis of pancreatic cancer patients.

High expression of olfactomedin-4 is correlated with chemoresistance and poor prognosis in pancreatic cancer.

Pancreatic cancer has an extremely poor prognosis, and identification of novel predictors of therapeutic efficacy and prognosis is urgently needed. Chemoresistance-related molecules are correlated with poor prognosis and may be effective targets for cancer treatment. Here, we aimed to identify novel molecules correlated with chemoresistance and poor prognosis in pancreatic cancer. We established 10 patient-derived xenograft (PDX) lines from patients with pancreatic cancer and performed next-generation sequencing (NGS) of tumor tissues from PDXs after treatment with standard drugs. We established a gene-transferred tumor cell line to express chemoresistance-related molecules and analyzed the chemoresistance of the established cell line against standard drugs. Finally, we performed immunohistochemical (IHC) analysis of chemoresistance-related molecules using 80 pancreatic cancer tissues. From NGS analysis, we identified olfactomedin-4 (OLFM4) as having high expression in the PDX group treated with anticancer drugs. In IHC analysis, OLFM4 expression was also high in PDXs administered anticancer drugs compared with that in untreated PDXs. Chemoresistance was observed by in vitro analysis of tumor cell lines with forced expression of OLFM4. In an assessment of tissue specimens from 80 patients with pancreatic cancer, Kaplan-Meier analysis showed that patients in the low OLFM4 expression group had a better survival rate than patients in the high OLFM4 expression group. Additionally, multivariate analysis showed that high expression of OLFM4 was an independent prognostic factor predicting poor outcomes. Overall, our study revealed that high expression of OLFM4 was involved in chemoresistance and was an independent prognostic factor in pancreatic cancer. OLFM4 may be a candidate therapeutic target in pancreatic cancer.

Identification of a novel HLA-A24-restricted cytotoxic T lymphocyte epitope peptide derived from mesothelin in pancreatic cancer.

Pancreatic cancer involves highly malignant tumors, and the development of new therapeutic strategies is critical. Mesothelin is overexpressed in infiltrating pancreatic cancer cells and plays an important role in the invasion and migration processes. In this study, we focused on mesothelin as a tumor-specific antigen target for a pancreatic cancer vaccine. We first investigated the mesothelin-derived epitope peptide restricted to HLA-A*2402. A total of 19 candidate peptides were synthesized, and we then determined their potential to induce peptide-specific cytotoxic T lymphocytes (CTLs). Peptide-specific CTLs were induced by five peptides derived from mesothelin, and these CTLs successfully exhibited peptide-specific IFN-γ production. After the expansion of each CTL, two CTL lines were established, which were induced by mesothelin-10-5 peptide (AFYPGYLCSL). These CTL lines exhibited peptide-specific cytotoxicity and IFN-γ production. Moreover, we were able to generate mesothelin-10-5 peptide-specific CTL clones. These CTL clones also had specific cytotoxic activity against HLA-A*2402-positive pancreatic cancer cells that endogenously expressed mesothelin. These results indicate that the mesothelin-10-5 peptide is a novel HLA-A*2402 restricted CTL epitope and that it is a promising candidate target for antigen-specific immunotherapy against pancreatic cancers.

CD49d is a disease progression biomarker and a potential target for immunotherapy in Duchenne muscular dystrophy.

BACKGROUND: Duchenne muscular dystrophy (DMD) is caused by mutations in the dystrophin gene. The immune inflammatory response also contributes to disease progression in DMD patients. In a previous study, we demonstrated higher levels of circulating CD49dhi and CD49ehi T cells in DMD patients compared to healthy control. DMD patients are clinically heterogeneous and the functional defect cannot be correlated with genotype. Therefore, it is important to be able to define reliable noninvasive biomarkers to better define the disease progression at the beginning of clinical trials. RESULTS: We studied 75 DMD patients at different stages of their disease and observed that increased percentages of circulating CD4(+)CD49d(hi) and CD8(+)CD49d(hi) T lymphocytes were correlated with both severity and a more rapid progression of the disease. Moreover, T(+)CD49d(+) cells were also found in muscular inflammatory infiltrates. Functionally, T cells from severely affected patients exhibited higher transendothelial and fibronectin-driven migratory responses and increased adhesion to myotubes, when compared to control individuals. These responses could be blocked with an anti-CD49d monoclonal antibody. CONCLUSION: CD49d can be used as a novel biomarker to stratify DMD patients by predicting disease progression for clinical trials. Moreover, anti-CD49d peptides or antibodies can be used as a therapeutic approach to decrease inflammation-mediated tissue damage in DMD.

Predictive markers of clinical outcome in the GRMD dog model of Duchenne muscular dystrophy.

In the translational process of developing innovative therapies for DMD (Duchenne muscular dystrophy), the last preclinical validation step is often carried out in the most relevant animal model of this human disease, namely the GRMD (Golden Retriever muscular dystrophy) dog. The disease in GRMD dogs mimics human DMD in many aspects, including the inter-individual heterogeneity. This last point can be seen as a drawback for an animal model but is inherently related to the disease in GRMD dogs closely resembling that of individuals with DMD. In order to improve the management of this inter-individual heterogeneity, we have screened a combination of biomarkers in sixty-one 2-month-old GRMD dogs at the onset of the disease and a posteriori we addressed their predictive value on the severity of the disease. Three non-invasive biomarkers obtained at early stages of the disease were found to be highly predictive for the loss of ambulation before 6 months of age. An elevation in the number of circulating CD4(+)CD49d(hi) T cells and a decreased stride frequency resulting in a reduced spontaneous speed were found to be strongly associated with the severe clinical form of the disease. These factors can be used as predictive tests to screen dogs to separate them into groups with slow or fast disease progression before their inclusion into a therapeutic preclinical trial, and therefore improve the reliability and translational value of the trials carried out on this invaluable large animal model. These same biomarkers have also been described to be predictive for the time to loss of ambulation in boys with DMD, strengthening the relevance of GRMD dogs as preclinical models of this devastating muscle disease.

Myoblasts and embryonic stem cells differentially engraft in a mouse model of genetic dilated cardiomyopathy.

The functional and architectural benefits of embryonic stem cells (ESC) and myoblasts (Mb) transplantations into infarcted myocardium have been investigated extensively. Whereas ESC repopulated fibrotic areas and contributed to myocardial regeneration, Mb exerted their effects through paracrine secretions and scar remodeling. This therapeutic perspective, however, has been less explored in the setting of nonischemic dilated cardiomyopathies (DCMs). Our aim was to compare the integration and functional efficacy of ESC committed to cardiac fate by bone morphogenic protein 2 (BMP-2) pretreatment and Mb used as gold standard following their transplantation into the myocardium of a mouse model of laminopathy exhibiting a progressive and lethal DCM. After 4 and 8 weeks of transplantation, stabilization was observed in Mb-transplanted mice (P = 0.008) but not in groups of ESC-transplanted or medium-injected animals, where the left ventricular fractional shortening (LVFS) decreased by 32 ± 8% and 41 ± 8% respectively. Engrafted differentiated cells were consistently detected in myocardia of mice receiving Mb, whereas few or no cells were detected in the hearts of mice receiving ESC, except in two cases where teratomas were formed. These data suggest that committed ESC fail to integrate in DCM where scar tissue is absent to provide the appropriate niche, whereas the functional benefits of Mb transplantation might extend to nonischemic cardiomyopathy.

Establishment of bipotent progenitor cell clone from rat skeletal muscle.

The present study describes the isolation, cloning and characterization of adipogenic progenitor cells from rat skeletal muscle. Among the obtained 10 clones, the most highly adipogenic progenitor, 2G11 cells, were further characterized. In addition to their adipogenicity, 2G11 cells retain myogenic potential as revealed by formation of multinucleated myotubes when co-cultured with myoblasts. 2G11 cells were resistant to an inhibitory effect of basic fibroblast growth factor on adipogenesis, while adipogenesis of widely used preadipogenic cell line, 3T3-L1 cells, was suppressed almost completely by the same treatment. In vivo transplantation experiments revealed that 2G11 cells are able to possess both adipogenicity and myogenicity in vivo. These results indicate the presence of bipotent progenitor cells in rat skeletal muscle, and suggest that such cells may contribute to ectopic fat formation in skeletal muscle.

Reprogramming efficiency and quality of induced Pluripotent Stem Cells (iPSCs) generated from muscle-derived fibroblasts of mdx mice at different ages.

Induced pluripotent stem cells (iPSCs) hold promise as a potential treatment for Duchenne muscular dystrophy (DMD). To determine the impact of the donor's age on reprogramming, we generated iPSCs from muscle-derived fibroblasts (MuFs) of mdx mice aged 6 weeks, 6 months, and 14 months. MuFs from 14-month-old mdx mice showed lower proliferative activity and lower reprogramming efficiency, compared with those from younger mdx mice. Furthermore, iPSCs derived from 14-month-old mdx mice (14m-MuF-iPSCs) gradually lost Nanog expression, and regressed in conventional ES medium during passages. Interestingly, inhibition of TGF-ß signaling and BMP signaling stabilized Nanog expression and promoted self-renewal of 14m-MuF-iPSCs. Finally, rescued mdx-derived iPSCs efficiently differentiated into the skeletal muscle lineage.

Six family genes control the proliferation and differentiation of muscle satellite cells.

Muscle satellite cells are essential for muscle growth and regeneration and their morphology, behavior and gene expression have been extensively studied. However, the mechanisms involved in their proliferation and differentiation remain elusive. Six1 and Six4 proteins were expressed in the nuclei of myofibers of adult mice and the numbers of myoblasts positive for Six1 and Six4 increased during regeneration of skeletal muscles. Six1 and Six4 were expressed in quiescent, activated and differentiated muscle satellite cells isolated from adult skeletal muscle. Overexpression of Six4 and Six5 repressed the proliferation and differentiation of satellite cells. Conversely, knockdown of Six5 resulted in augmented proliferation, and that of Six4 inhibited differentiation. Muscle satellite cells isolated from Six4(+/-)Six5(-/-) mice proliferated to higher cell density though their differentiation was not altered. Meanwhile, overproduction of Six1 repressed proliferation and promoted differentiation of satellite cells. In addition, Six4 and Six5 repressed, while Six1 activated myogenin expression, suggesting that the differential regulation of myogenin expression is responsible for the differential effects of Six genes. The results indicated the involvement of Six genes in the behavior of satellite cells and identified Six genes as potential target for manipulation of proliferation and differentiation of muscle satellite cells for therapeutic applications.

Muscle CD31(-) CD45(-) side population cells promote muscle regeneration by stimulating proliferation and migration of myoblasts.

CD31(-) CD45(-) side population (SP) cells are a minor SP subfraction that have mesenchymal stem cell-like properties in uninjured skeletal muscle but that can expand on muscle injury. To clarify the role of these SP cells in muscle regeneration, we injected green fluorescent protein (GFP)-positive myoblasts with or without CD31(-) CD45(-) SP cells into the tibialis anterior muscles of immunodeficient NOD/scid mice or dystrophin-deficient mdx mice. More GFP-positive fibers were formed after co-transplantation than after transplantation of GFP-positive myoblasts alone in both mdx and NOD/scid muscles. Moreover, grafted myoblasts were more widely distributed after co-transplantation than after transplantation of myoblasts alone. Immunohistochemistry with anti-phosphorylated histone H3 antibody revealed that CD31(-) CD45(-) SP cells stimulated cell division of co-grafted myoblasts. Genome-wide gene expression analyses showed that these SP cells specifically express a variety of extracellular matrix proteins, membrane proteins, and cytokines. We also found that they express high levels of matrix metalloproteinase-2 mRNA and gelatinase activity. Furthermore, matrix metalloproteinase-2 derived from CD31(-) CD45(-) SP cells promoted migration of myoblasts in vivo. Our results suggest that CD31(-) CD45(-) SP cells support muscle regeneration by promoting proliferation and migration of myoblasts. Future studies to further define the molecular and cellular mechanisms of muscle regeneration will aid in the development of cell therapies for muscular dystrophy.

18alpha-glycyrrhetinic acid induces phenotypic changes of skeletal muscle cells to enter adipogenesis.

The importance of connexins is implicated in proliferation and differentiation of cells. In skeletal muscle cells, connexin43 (Cx43) has been identified as the major connexin, and gap-junctional communication mediated by connexins has been shown to be required for their myogenic differentiation. In addition, inhibition of connexin function has been shown to induce transdifferentiation of osteoblasts to an adipocytic phenotype. In the present study, we examined whether the inhibition of connexin function could induce phenotypic changes in skeletal muscle cells. Treatment of skeletal muscle cells with an inhibitor of connexin function, 18alpha-glycyrrhetinic acid (AGRA), resulted in a reduction in the number of MyoD-positive cells and complete inhibition of myotube formation, concomitantly with an increase in the number of C/EBPalpha-positive cells. AGRA-treated cells cultured in adipogenic differentiation medium could give rise to mature adipocytes that express both PPARgamma and C/EBPalpha. The presence of AGRA during adipogenic differentiation did not inhibit adipogenesis of skeletal muscle cells. AGRA treatment did not affect Cx43 expression in skeletal muscle cells but reduced its phosphorylation. These results indicate that inhibition of connexin function induces phenotypic changes of skeletal muscle cells to enter adipogenesis.

Adipogenic potential of satellite cells from distinct skeletal muscle origins in the rat.

The possible relationship between myofiber type composition and adipose tissue development in skeletal muscle in vivo has been suggested. Recent evidence indicated that satellite cells are multipotent cells that can undergo not only myogenic, but also adipogenic differentiation. In the present study, rat satellite cells were isolated from soleus, back, extensor digitorum longus, tibialis anterior and quadriceps muscles, and their adipogenic potentials were compared by culturing them under adipogenic conditions in vitro. Cells from soleus muscle exhibited the highest adipogenic potential as judged from Oil Red-staining and immunocytochemical C/EBPalpha-staining. The adipogenic potential of satellite cells was positively correlated with type I myofiber distribution in the corresponding muscle of origin. These results demonstrated that the adipogenic potential of satellite cells differs according to the muscle of origin and suggested that its possible correlation to type I myofiber distribution may account for preferential adipose tissue development in slow oxidative muscles.

Increased adipogenicity of cells from regenerating skeletal muscle.

Adipose tissue development is observed in some muscle pathologies, however, mechanisms that induce accumulation of this tissue as well as its cellular origin are unknown. The adipogenicity of cells from bupivacaine hydrochloride (BPVC)-treated and untreated muscle was compared in vitro. Culturing cells from both BPVC-treated and untreated muscles in adipogenic differentiation medium (ADM) for 10 days resulted in the appearance of mature adipocytes, but their number was 3.5-fold higher in cells from BPVC-treated muscle. Temporal expressions of PPARgamma and the presence of lipid droplets during adipogenic differentiation were examined. On day 2 of culture in ADM, only cells from BPVC-treated muscle were positive both for PPARgamma and lipid droplets. Pref-1 was expressed in cells from untreated muscle, whereas its expression was absent in cells from BPVC-treated muscle. In ADM, the presence of insulin, which negates an inhibitory effect of Pref-1 on adipogenic differentiation, was required for PPARgamma2 expression in cells from untreated muscle, but not for cells from BPVC-treated muscle. These results indicate that BPVC-induced degenerative/regenerative changes in muscle lead to increased adipogenicity of cells, and suggest that this increased adipogenicity not only involves an increase in the number of cells having adipogenic potential, but also contributes to the progression of these cells toward adipogenic differentiation.

Analyses of hind leg skeletons in human growth hormone transgenic rats.

Growth hormone (GH) is essential in the development and growth of the skeleton and for the maintenance of bone mass and density, and its secretion is known to decline with aging. We have previously produced transgenic rats with low circulating GH that represent several age-associated phenotypes such as obesity, insulin-resistance and leptin-resistance. In the present study, the cross-sectional area, bone mineral density, and strength indexes of the hind leg skeletons of the transgenic rats were examined by an X-ray computed tomography scanning. The mean cross-sectional area of the transgenic rats showed no increase after 2 months old up to 8 months old and the strength indexes were significantly lower than their non-transgenic siblings at all ages examined. The trabecular bone mineral density in the transgenic rats drastically decreased at 8 months old, while the cortical bone mineral density was comparable to the non-transgenic rats, suggesting the onset of osteoporosis at this period. The results obtained in this study indicate that the transgenic rats could be useful model to gain insight into the complex mechanism leading to osteoporosis with aging.