Current Issue : January - March Volume : 2019 Issue Number : 1 Articles : 5 Articles
Based on its ability to express high levels of protein, baculovirus has been widely used for\nrecombinant protein production in insect cells for more than thirty years with continued technical\nimprovements. In addition, baculovirus has been successfully applied for foreign gene delivery into\nmammalian cells without any viral replication. However, several CpG motifs are present throughout\nbaculoviral DNA and induce an antiviral response in mammalian cells, resulting in the production\nof pro-inflammatory cytokines and type I interferon through a Toll-like receptor (TLR)-dependent\nor -independent signaling pathway, and ultimately limiting the efficiency of transgene expression.\nOn the other hand, by taking advantage of this strong adjuvant activity, recombinant baculoviruses\nencoding neutralization epitopes can elicit protective immunity in mice. Moreover, immunodeficient\ncells, such as hepatitis C virus (HCV)- or human immunodeficiency virus (HIV)-infected cells,\nare more susceptible to baculovirus infection than normal cells and are selectively eliminated by the\napoptosis-inducible recombinant baculovirus. Here, we summarize the application of baculovirus\nas a gene expression vector and the mechanism of the host innate immune response induced by\nbaculovirus in mammalian cells. We also discuss the future prospects of baculovirus vectors....
Ciliary neurotrophic factor (CNTF) promotes survival and enhances long-distance regeneration of injured axons in parts of the\nadult CNS. Here we tested whether CNTF gene therapy targeting corticospinal neurons (CSN) in motor-related regions of the\ncerebral cortex promotes plasticity and regrowth of axons projecting into the female adult F344 rat spinal cord after moderate\nthoracic (T10) contusion injury (SCI). Cortical neurons were transduced with a bicistronic adeno-associated viral vector (AAV1)\nexpressing a secretory form of CNTF coupled to mCHERRY (AAV-CNTFmCherry) or with control AAV only (AAV-GFP) two\nweeks prior to SCI. In some animals, viable or nonviable F344 rat mesenchymal precursor cells (rMPCs) were injected into the\nlesion site two weeks after SCI to modulate the inhibitory environment. Treatment with AAV-CNTFmCherry, as well as with\nAAV-CNTFmCherry combined with rMPCs, yielded functional improvements over AAV-GFP alone, as assessed by open-field\nand Ladderwalk analyses. Cyst size was significantly reduced in the AAV-CNTFmCherry plus viable rMPC treatment group.\nCortical injections of biotinylated dextran amine (BDA) revealed more BDA-stained axons rostral and alongside cysts in the\nAAV-CNTFmCherry versus AAV-GFP groups. After AAV-CNTFmCherry treatments, many sprouting mCherry-immunopositive\naxons were seen rostral to the SCI, and axons were also occasionally found caudal to the injury site. These data suggest that\nCNTF has the potential to enhance corticospinal repair by transducing parent CNS populations....
MicroRNAs (miRNAs) regulate gene expression at posttranscriptional level by triggering\nRNA interference. In such a sense, aberrant expressions of miRNAs play critical roles in the\npathogenesis of many disorders, including Parkinsonâ??s disease (PD). Controlling the level of specific\nmiRNAs in the brain is thus a promising therapeutic strategy for neuroprotection. A fundamental\nneed for miRNA regulation (either replacing or inhibition) is a carrier capable of delivering\noligonucleotides into brain cells. This study aimed to examine a polymeric magnetic particle,\nNeuromag®, for delivery of synthetic miRNA inhibitors in the rat central nervous system. We injected\nthe miRNA inhibitor complexed with Neuromag® into the lateral ventricles next to the striatum,\nby stereotaxic surgery. Neuromag efficiently delivered oligonucleotides in the striatum and\nseptum areas, as shown by microscopy imaging of fluorescein isothiocyanate (FITC)-labeled oligos\nin astrocytes and neurons. Transfected oligos showed efficacy concerning miRNA inhibition.\nNeuromag®-structured miR-134 antimiR (0.36 nmol) caused a significant 0.35 fold decrease of striatal\nmiR-134, as revealed by real-time quantitative polymerase chain reaction (RT-qPCR). In conclusion,\nthe polymeric magnetic particle Neuromag® efficiently delivered functional miRNA inhibitors in\nbrain regions surrounding lateral ventricles, particularly the striatum. This delivery system holds\npotential as a promising miRNA-based disease-modifying drug and merits further pre-clinical studies\nusing animal models of PD....
The development of precise and modulated methods for customized manipulation of DNA is an important objective for the study\nand engineering of biological processes and is essential for the optimization of gene therapy, metabolic flux, and synthetic gene\nnetworks. The clustered regularly interspaced short palindromic repeat- (CRISPR-) associated protein 9 is an RNA-guided\nsite-specific DNA-binding complex that can be reprogrammed to specifically interact with a desired DNA sequence target.\nCRISPR-Cas9 has been used in a wide variety of applications ranging from basic science to the clinic, such as gene\ntherapy, gene regulation, modifying epigenomes, and imaging chromosomes. Although Cas9 has been successfully used as a\nprecise tool in all these applications, some limitations have also been reported, for instance (i) a strict dependence on a\nprotospacer-adjacent motif (PAM) sequence, (ii) aberrant off-target activity, (iii) the large size of Cas9 is problematic for\nCRISPR delivery, and (iv) lack of modulation of protein binding and endonuclease activity, which is crucial for precise\nspatiotemporal control of gene expression or genome editing. These obstacles hinder the use of CRISPR for disease treatment\nand in wider biotechnological applications. Protein-engineering approaches offer solutions to overcome the limitations of Cas9\nand generate robust and efficient tools for customized DNA manipulation. Here, recent protein-engineering approaches for\nexpanding the versatility of the Streptococcus pyogenes Cas9 (SpCas9) is reviewed, with an emphasis on studies that improve or\ndevelop novel protein functions through domain fusion or splitting, rational design, and directed evolution....
Recombinant viruses are novel therapeutic agents that can be utilized for treatment of\nvarious diseases, including cancers. Recombinant viruses can be engineered to express foreign\ntransgenes and have a broad tropism allowing gene expression in a wide range of host cells. They can\nbe selected or designed for specific therapeutic goals; for example, recombinant viruses could be\nused to stimulate host immune response against tumor-specific antigens and therefore overcome\nthe ability of the tumor to evade the hostâ??s immune surveillance. Alternatively, recombinant viruses\ncould express immunomodulatory genes which stimulate an anti-cancer immune response. Oncolytic\nviruses can replicate specifically in tumor cells and induce toxic effects leading to cell lysis and\napoptosis. However, each of these approaches face certain difficulties that must be resolved to achieve\nmaximum therapeutic efficacy. In this review we discuss actively developing approaches for cancer\ntherapy based on recombinant viruses, problems that need to be overcome, and possible prospects\nfor further development of recombinant virus based therapy...
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