Molecular Therapy最新文献

Despite various available treatments, highly prevalent osteoarthritis cannot be cured in patients. In light of evidence showing mitochondria dysfunction during the disease progression, our goal was to develop a novel therapeutic concept based on the transplantation of mitochondria as platforms to deliver recombinant adeno-associated viral (rAAV) gene vectors with a potency for osteoarthritis. For the first time to our best knowledge, we report the successful creation of a safe mitochondria/rAAV system effectively promoting the overexpression of a candidate insulin-like growth factor I (IGF-I) by administration to autologous human osteoarthritic articular chondrocytes versus control conditions (reporter mitochondria/rAAV lacZ system, rAAV-free system, absence of mitochondria transplantation) (up to 8.4-fold difference). The candidate mitochondria/rAAV IGF-I system significantly improved key activities in the transplanted cells (proliferation/survival, extracellular matrix production, mitochondria functions) relative to the control conditions (up to 9.5-fold difference), including when provided in a PF127 hydrogel for reinforced delivery (up to 5.9-fold difference). Such effects were accompanied with increased levels of cartilage-specific SOX9 and Mfn-1 (mitochondria fusion) and with decreased levels of Drp-1 (mitochondria fission) and proinflammatory TNF-α (up to 4.5-fold difference). This study shows the potential of combining the use of mitochondria with rAAV as a promising approach for human OA.

Sepsis-induced thrombocytopenia (SIT) is a widely accepted predictor of poor prognosis during sepsis, while the mechanism of SIT remains elusive. In this study, we revealed that SIT patients and septic mice exhibited higher levels of pro-inflammatory macrophages and phosphorylated Bruton's tyrosine kinase (p-BTK) expression in macrophages, which were closely correlated with platelet counts. Treatment with the BTK inhibitor BGB-3111 in SIT mice resulted in enhanced production of megakaryocytes and platelets. Depletion of macrophages in SIT mice and coculture experiments further confirmed the critical role of macrophages in the improvement of platelet count induced by BGB-3111. By performing single-cell RNA sequencing on bone marrow-derived cells from SIT mice, we not only confirmed the connection between macrophages and megakaryocytes influenced by BTK but also identified a potential mediation through the Rap1 signaling pathway in macrophages. Subsequent experiments in macrophages demonstrated that inhibition of BTK signaling impeded the pro-inflammatory polarization of macrophages by targeting the Rap1/NF-κB signaling pathway. In conclusion, our study highlights the crucial role of macrophages in SIT, and inhibiting phosphorylation of BTK in macrophages may alleviate SIT through the Rap1/NF-κB signaling pathway.

Gene therapy achieves therapeutic benefits by delivering genetic materials, packaged within a delivery vehicle, to target cells with defective genes. This approach has shown promise in treating various conditions, including cancer, metabolic disorders, and tissue-degenerative diseases. Over the past 5 years, molecular imaging has increasingly supported gene therapy development in both preclinical and clinical studies. High-quality images from positron emission tomography (PET), single-photon emission computed tomography (SPECT), magnetic resonance imaging (MRI), and computed tomography (CT) enable quantitative and reliable monitoring of gene therapy. Most reported studies have applied imaging biomarkers to non-invasively evaluate the outcomes of gene therapy. This review aims to inform researchers in molecular imaging and gene therapy about the integration of these two disciplines. We highlight recent developments in using imaging biomarkers to monitor the outcome of in vivo gene therapy, where the therapeutic delivery vehicle is administered systemically. In addition, we discuss prospects for further incorporating imaging biomarkers to support the development and application of gene therapy.

Oncolytic herpes simplex viruses (oHSV) preferentially replicate in cancer cells while inducing antitumor immunity, and thus, they are often referred to as in situ cancer vaccines. OHSV infection of tumors elicits diverse host immune responses comprising both innate and adaptive components. Although the innate and adaptive immune responses primarily target the tumor, they also contribute to antiviral immunity, limiting viral replication/oncolysis. OHSV-encoded proteins use various mechanisms to evade host antiviral pathways and immune recognition, favoring oHSV replication, oncolysis, and spread. In general, oHSV infection and replication within tumors results in a series of sequential events, such as oncolysis and release of tumor and viral antigens, dendritic cell-mediated antigen presentation, T cell priming and activation, T cell trafficking and infiltration to tumors, and T cell recognition of cancer cells, leading to tumor (and viral) clearance. These sequential events align with all steps of the cancer-immunity cycle. However, a comprehensive understanding of the interplay between oHSV and host immune responses is crucial to optimize oHSV-induced antitumor immunity and efficacy. Therefore, this review aims to elucidate oHSV's communication with innate and adaptive immune systems and use such interactions to improve oHSV's potential as a potent immunovirotherapeutic agent against cancer.

B cells are an attractive platform for engineering to produce protein-based biologics absent in genetic disorders, and potentially for the treatment of metabolic diseases and cancer. As part of pre-clinical development of B cell medicines, we demonstrate a method to collect, ex vivo expand, differentiate, radioactively label, and track adoptively transferred non-human primate (NHP) B cells. These cells underwent 10- to 15-fold expansion, initiated IgG class switching, and differentiated into antibody-secreting cells. Zirconium-89-oxine-labeled cells were infused into autologous donors without any preconditioning and tracked by PET/CT imaging. Within 24 h of infusion, 20% of the initial dose homed to the bone marrow and spleen and distributed stably and equally between the two. Interestingly, approximately half of the dose homed to the liver. Image analysis of the bone marrow demonstrated inhomogeneous distribution of the cells. The subjects experienced no clinically significant side effects or laboratory abnormalities. A second infusion of B cells into one of the subjects resulted in an almost identical distribution of cells, suggesting possibly a non-limiting engraftment niche and feasibility of repeated infusions. This work supports the NHP as a valuable model to assess the potential of B cell medicines as potential treatment for human diseases.

Enhancing protective immunity in the respiratory tract is crucial to combat influenza infection and transmission. Developing mucosal universal influenza vaccines requires effective delivery platforms to overcome the respiratory mucosal barrier and stimulate appropriate innate immune reactions, thereby bridging adaptive immune responses with minimal necessary inflammation. Meanwhile, the vaccine platforms must be biocompatible. This study employed cell-derived extracellular vesicles (EVs) as a mucosal universal influenza vaccine platform. By conjugating influenza hemagglutinin (HA) onto EV surfaces through HA-receptor interaction, we achieved an upside-down (inverted) influenza HA configuration that exposed the conserved HA stalk region while partially hiding the globular head domain. Intranasal immunization with the resulting EVs induced robust HA stalk- and virus-specific serum antibody and mucosal immune responses in mice, protecting against heterologous virus infection. Notably, EVs derived from the lung epithelial cell line A549 induced superior cross-reactive antibodies and enhanced protection upon intranasal immunization. EVs conjugating multivalent HA elicited broadly cross-reactive antibody and cellular responses against different influenza strains. Our results demonstrated that EVs conjugating multiple inverted HAs represented an effective strategy for developing a mucosal universal influenza vaccine.

Proliferation of vascular smooth muscle cells (vSMCs) is a crucial contributor to pathological vascular remodeling. MicroRNAs (miRNAs) are powerful gene regulators and attractive therapeutic agents. Here, we aimed to systematically identify and characterize miRNAs with therapeutic potential in targeting vSMC proliferation. Using high-throughput screening, we assessed the impact of 2,042 human miRNA mimics on vSMC proliferation and identified seven miRNAs with novel vSMC anti-proliferative function: miR-323a-3p, miR-449b-5p, miR-491-3p, miR-892b, miR-1827, miR-4774-3p, and miR-5681b. miRNA-mimic treatment affects proliferation of vSMCs from different vascular beds. Focusing on vein graft failure, where miRNA-based therapeutics can be applied to the graft ex vivo, we showed that these miRNAs reduced human saphenous vein smooth muscle cell (HSVSMC) proliferation without toxic effect. HSVSMC transcriptomics revealed a distinct set of targets for each miRNA, leading to the common downregulation of a cell-cycle gene network for all miRNAs. For miR-449b-5p, we showed that its candidate target, CCND1, contributes to HSVSMC proliferation. In contrast to HSVSMCs, miRNA overexpression in endothelial cells led to a limited response in terms of proliferation and transcriptomics. In an ex vivo vein organ model, overexpression of miR-323a-3p and miR-449b-5p reduced medial proliferation. Collectively, the results of our study show the therapeutic potential of seven miRNAs to target pathological vascular remodeling.

Painful diabetic neuropathy commonly affects the peripheral nervous system in individuals with diabetes. However, the pathological processes and mechanisms underlying diabetic neuropathic pain remain unclear. We aimed to identify the overall profiles and screen for genes potentially involved in pain mechanisms using transcriptome analysis of the dorsal root ganglion of diabetic mice treated with streptozotocin (STZ). Using RNA sequencing, we identified differentially expressed genes between streptozotocin-treated diabetic mice and controls, focusing on altered GABAergic neuron-related genes and inflammatory pathways. Behavioral and molecular analyses revealed a marked reduction in GABAergic neuronal markers (GAD65, GAD67, VGAT) and increased pro-inflammatory cytokines (TNF-α, IL-1β, IL-6) in the diabetic group compared with controls. Intrathecal administration of lentiviral vectors expressing transcription factors Ascl1 and Lhx6 reversed pain hypersensitivity and restored normal expression of GABAergic genes and inflammatory mediators. Protein-protein interaction network analysis revealed five key proteins influenced by Ascl1 and Lhx6 treatment, including those in the JunD/FosB/C-fos signaling pathway. These findings suggest that Ascl1 and Lhx6 mitigate diabetic neuropathic pain by modulating GABAergic neuronal function, pro-inflammatory responses, and pain-related channels (TRPV1, Nav1.7). These results provide a basis for developing transcription factor-based therapies targeting GABAergic neurons for diabetic neuropathic pain relief.

Loss-of-function mutations in the chromodomain helicase DNA-binding 8 (CHD8) gene are strongly associated with autism spectrum disorders (ASDs). Indeed, the reduction of CHD8 causes transcriptional, epigenetic, and cellular phenotypic changes correlated to disease, which can be monitored in assessing new therapeutic approaches. SINEUPs are a functional class of natural and synthetic antisense long non-coding RNAs able to stimulate the translation of sense target mRNA, with no effect on transcription. Here, we employed synthetic SINEUP-CHD8 targeting the first and third AUG of the CHD8 coding sequence to efficiently stimulate endogenous CHD8 protein production. SINEUP-CHD8 were effective in cells with reduced levels of the target protein and in patient-derived fibroblasts with CHD8 mutations. Functionally, SINEUP-CHD8 were able to revert molecular phenotypes associated with CHD8 suppression, i.e., genome-wide transcriptional dysregulation, and the reduction of H3K36me3 levels. Strikingly, in chd8-morpholino-treated and ENU mutant zebrafish embryos, SINEUP-chd8 injection confirmed the ability of SINEUP RNA to rescue the chd8-suppression-induced macrocephaly phenotype and neuronal hyperproliferation. Thus, SINEUP-CHD8 molecule(s) represent a proof-of-concept toward the development of an RNA-based therapy for neurodevelopmental syndromes with implications for, and beyond ASD, and relevant to genetic disorders caused by protein haploinsufficiency.

Self-amplifying RNA (saRNA) vectors are a next-generation RNA technology that extends the expression of heterologous genes. Clinical trials have shown the dose-sparing capacity of saRNA vectors in a vaccine context compared with conventional messenger RNA. However, saRNA vectors have historically been based on a limited number of alphaviruses, and only the Venezuelan equine encephalitis virus-based saRNA vaccines have been used clinically. Here, we designed genotypically distinct alphaviral saRNA vectors and characterized their performance in mammalian cell lines, human skin explants and mice. Five of the 12 vectors had substantial luciferase expression in mice with variable pharmacokinetics, enabling modulation of both the magnitude and duration of protein expression. Additionally, we demonstrated that the alphaviral genotype of the saRNA significantly impacts the immunogenicity of saRNA vaccines, including the humoral and cellular responses in mice. Given the differences in RNA reactogenicity and expression between mice and humans, we assessed the saRNA vectors in human skin explants obtained from patients and observed high transgene expression. saRNA bioluminescence and immunogenicity in different mice strains were highly correlative, while minimal correlation was observed when compared with human explants and mammalian cell lines. This work demonstrates that efficacious saRNA vaccines and therapies can be produced by adapting genetically diverse alphaviruses into vectors.