Integrated mRNA-miRNA transcriptome profiling of blood immune responses potentially related to pulmonary fibrosis in forest musk deer.

Background: Forest musk deer (FMD, Moschus Berezovskii) is a critically endangered species world-widely, the death of which can be caused by pulmonary disease in the farm. Pulmonary fibrosis (PF) was a huge threat to the health and survival of captive FMD. MicroRNAs (miRNAs) and messenger RNAs (mRNAs) have been involved in the regulation of immune genes and disease development. However, the regulatory profiles of mRNAs and miRNAs involved in immune regulation of FMD are unclear. Methods: In this study, mRNA-seq and miRNA-seq in blood were performed to constructed coexpression regulatory networks between PF and healthy groups of FMD. The hub immune- and apoptosis-related genes in the PF blood of FMD were explored through Gene Ontology (GO) and the Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis. Further, protein-protein interaction (PPI) network of immune-associated and apoptosis-associated key signaling pathways were constructed based on mRNA-miRNA in the PF blood of the FMD. Immune hub DEGs and immune hub DEmiRNAs were selected for experimental verification using RT-qPCR. Results: A total of 2744 differentially expressed genes (DEGs) and 356 differentially expressed miRNAs (DEmiRNAs) were identified in the PF blood group compared to the healthy blood group. Among them, 42 DEmiRNAs were negatively correlated with 20 immune DEGs from a total of 57 correlations. The DEGs were significantly associated with pathways related to CD molecules, immune disease, immune system, cytokine receptors, T cell receptor signaling pathway, Th1 and Th2 cell differentiation, cytokine-cytokine receptor interaction, intestinal immune network for IgA production, and NOD-like receptor signaling pathway. There were 240 immune-related DEGs, in which 186 immune-related DEGs were up-regulated and 54 immune-related DEGs were down-regulated. In the protein-protein interaction (PPI) analysis of immune-related signaling pathway, TYK2, TLR2, TLR4, IL18, CSF1, CXCL13, LCK, ITGB2, PIK3CB, HCK, CD40, CD86, CCL3, CCR7, IL2RA, TLR3, and IL4R were identified as the hub immune genes. The mRNA-miRNA coregulation analysis showed that let-7d, miR-324-3p, miR-760, miR-185, miR-149, miR-149-5p, and miR-1842-5p are key miRNAs that target DEGs involved in immune disease, immune system and immunoregulation. Conclusion: The development and occurrence of PF were significantly influenced by the immune-related and apoptosis-related genes present in PF blood. mRNAs and miRNAs associated with the development and occurrence of PF in the FMD.

Enhanced nuclear gene delivery via integrating and streamlining intracellular pathway.

The essential challenge of gene therapy is to develop safe and efficient vectors that escort genes to target sites. However, due to the cumbersome workflow of gene transfection into cells, successive gene loss occurs. This leads to considerable reductions in nuclear gene uptake, eventually causing low gene expression. Herein, we designed a gene vector named CA3S2 (C: N,N'-cystamine-bis-acrylamide [CBA], A: agmatine dihydrochloride [Agm], S: 4-(2-aminoethyl) benzenesulfonamide [ABS]) with excellent gene transfection ability. This vector can promote gene delivery to the nucleus via enhanced endoplasmic reticulum (ER) targeting through integrating and streamlining of the complex intracellular pathway. Briefly, ABS endowed CA3S2/DNA nanoparticles with not only a natural ER-targeting tendency attributed to the caveolae-mediated pathway but also direct receptor-binding capacity on the ER surface. Agm enabled CA3S2 to enhance lysosomal escape and nuclear uptake ability. The gene delivery efficiency of CA3S2 was significantly better than that of polyethyleneimine 25K (PEI 25K). Therefore, CA3S2 is a promising gene carrier, and the ER-targeting strategy involving intracellular pathway integration and streamlining has potential for gene therapy.

Gene therapy strategies for rare monogenic disorders with nuclear or mitochondrial gene mutations.

Rare monogenic disorders are a group of single-gene-mutated diseases that have a low incidence rate (less than 0.5‰) and eventually lead to patient disability and even death. Due to the relatively low number of people affected, these diseases typically fail to attract a great deal of commercial investment and research interest, and the affected patients thus have unmet medical needs. Advances in genomics biology, gene editing, and gene delivery can now offer potentially effective options for treating rare monogenic diseases. Herein, we review the application of gene therapy strategies (traditional gene therapy and gene editing) against various rare monogenic diseases with nuclear or mitochondrial gene mutations, including eye, central nervous system, pulmonary, systemic, and blood cell diseases. We summarize their pathologic features, address the barriers to gene delivery for these diseases, discuss available therapies in the clinic and in clinical trials, and sum up in-development gene delivery systems for various rare monogenic disorders. Finally, we elaborate the possible directions and outlook of gene therapy for rare monogenic disorders.

Pathologically Responsive Mitochondrial Gene Therapy in an Allotopic Expression-Independent Manner Cures Leber's Hereditary Optic Neuropathy.

Leber's hereditary optic neuropathy (LHON) is a rare inherited blindness caused by mutations in the mitochondrial DNA (mtDNA). The disorder is untreatable and tricky, as the existing chemotherapeutic agent Idebenone alleviates symptoms rather than overcoming the underlying cause. Although some studies have made progress on allotopic expression for LHON, in situ mitochondrial gene therapy remains challenging, which may simplify delivery procedures to be a promising therapeutic for LHON. LHON becomes more difficult to manage in the changed mitochondrial microenvironment, including increasing reactive oxygen species (ROS) and decreasing mitochondrial membrane potential (MMP). Herein, a pathologically responsive mitochondrial gene delivery vector named [triphenylphosphine-terminated poly(sulfur-containing thioketal undecafluorohexylamine histamine) and Ide-terminated poly(sulfur-containing thioketal undecafluorohexylamine histamine)] (TISUH) is reported to facilitate commendable in situ mitochondrial gene therapy for LHON. TISUH directly targets diseased mitochondria via triphenylphosphine and fluorination addressing the decreasing MMP. In addition, TISUH can be disassembled by high mitochondrial ROS levels to release functional genes for enhancing gene transfection efficiency and fundamentally correcting genetic abnormalities. In both traditional and gene-mutation-induced LHON mouse models, TISUH-mediated gene therapy shows satisfactory curative effect through the sustained therapeutic protein expression in vivo. This work proposes a novel pathologically responsive in situ mitochondrial delivery platform and provides a promising approach for refractory LHON as well as other mtDNA mutated diseases treatments.