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Tapping into the ability of engineered mesenchymal stem cells (MSCs) to mobilise into the tumour has expanded the scope of cancer treatment. Engineered MSCs expressing tumour necrosis factor (TNF)-related apoptosis inducing ligand (MSC-TRAIL) could serve as a platform for an efficient and targeted form of therapy. However, the presence of cancer stem cells (CSCs) that are resistant to TRAIL and apoptosis may represent a challenge for effective treatment. Nonetheless, with the discovery of small molecular inhibitors that could target CSCs and tumour signalling pathways, a higher efficacy of MSC-TRAIL mediated tumour inhibition can be achieved. This might pave the way for a more effective form of combined therapy, which leads to a better treatment outcome. In this review, we first discuss the tumour-homing capacity of MSCs, its effect in tumour tropism, the different approach behind genetically-engineered MSCs, and the efficacy and safety of each agent delivered by these MSCs. Then, we focus on how sensitisation of CSCs and tumours using small molecular inhibitors can increase the effect of these cells to either TRAIL or MSC-TRAIL mediated inhibition. In the conclusion, we address a few questions and safety concerns regarding the utilization of engineered MSCs for future treatment in patients.
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Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais/metabolismo , Neoplasias/terapia , Células-Tronco Neoplásicas , Ligante Indutor de Apoptose Relacionado a TNF/metabolismo , Animais , Terapia Baseada em Transplante de Células e Tecidos , Engenharia Genética , Humanos , Camundongos , Ligante Indutor de Apoptose Relacionado a TNF/genéticaRESUMO
MicroRNAs (miRNAs) are small noncoding RNA known to regulate brain development. The expression of two novel miRNAs, namely, miR-344b and miR-344c, was characterized during mouse brain developmental stages in this study. In situ hybridization analysis showed that miR-344b and miR-344c were expressed in the germinal layer during embryonic brain developmental stages. In contrast, miR-344b was not detectable in the adult brain while miR-344c was expressed exclusively in the adult olfactory bulb and cerebellar granular layer. Stem-loop RT-qPCR analysis of whole brain RNAs showed that expression of the miR-344b and miR-344c was increased as brain developed throughout the embryonic stage and maintained at adulthood. Further investigation showed that these miRNAs were expressed in adult organs, where miR-344b and miR-344c were highly expressed in pancreas and brain, respectively. Bioinformatics analysis suggested miR-344b and miR-344c targeted Olig2 and Otx2 mRNAs, respectively. However, luciferase experiments demonstrated that these miRNAs did not target Olig2 and Otx2 mRNAs. Further investigation on the locality of miR-344b and miR-344c showed that both miRNAs were localized in nuclei of immature neurons. In conclusion, miR-344b and miR-344c were expressed spatiotemporally during mouse brain developmental stages.
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Encéfalo/embriologia , Encéfalo/metabolismo , MicroRNAs/metabolismo , Neurônios/metabolismo , Animais , Feminino , Camundongos , Camundongos Endogâmicos C57BLRESUMO
A nano-sized polymer, dextran-spermine (D-SPM), was shown to have the capacity to deliver gene to the lung of mouse via intranasal route. In this study, assessments on the safety profile of D-SPM were performed to complement the gene expression results. African green monkey kidney fibroblast (COS-7) and human adenocarcinoma breast (MCF-7) cells transfected with D-SPM/pDNA showed massive reduction in the number of viable cells. As for in vivo study, elevated level of neutrophils was observed, despite the minimal level of pro-inflammatory cytokines (TNF-alpha, IL-12, IFN-gamma) detected in the bronchoalveolar lavage fluid (BALF) of mice treated with the D-SPM/pDNA complexes. Histology profile examinations of the lungs showed mild inflammatory responses, with inflamed areas overlap with healthy areas. Although reduction of mice weight was seen at day 1 post administration, the mice did not show any sign of abnormal behavior or physical appearance. Biodistribution study was performed to determine the ability of the D-SPM/pDNA complexes to infiltrate to other non-intended organs. The result showed that the D-SPM/pDNA complexes were only localized at the lung and no gene expression was detected in other organs or blood. In short, these results indicate that the D-SPM/pDNA exhibited mild toxicity in the mouse lungs.
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Dextrinas/administração & dosagem , Vetores Genéticos/efeitos adversos , Pulmão/metabolismo , Espermina/administração & dosagem , Animais , Líquido da Lavagem Broncoalveolar , Células COS , Chlorocebus aethiops , Dextrinas/farmacocinética , Ensaio de Imunoadsorção Enzimática , Feminino , Células MCF-7 , Camundongos , Camundongos Endogâmicos BALB C , Plasmídeos , Espermina/farmacocinética , Distribuição TecidualRESUMO
An embryonic stem cell (ESC) is a good tool to generate neurons in vitro and can be used to mimic neural development in vivo. It has been widely used in research to examine the role of cell signalling during neuronal development, test the effects of drugs on neurons, and generate a large population of functional neurons. So far, a number of protocols have been established to promote the differentiation of ESCs, such as direct and indirect differentiation. One of the widely used protocols to generate neurons is through the spontaneous formation of multicellular aggregates known as embryonic bodies (EBs). However, for some, it is not clear why EB protocol could be the protocol of choice. EB also is known to mimic an early embryo; hence, knowing the similarities between EB and an early embryo is essential, particularly the information on the players that promote the formation of EBs or the aggregation of ESCs. This review paper focuses on these issues and discusses further the generation of neural cells from EBs using a well-known protocol, the 4-/4+ protocol.
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Spinal muscular atrophy (SMA) is an autosomal recessive neuromuscular disorder due to deletion or mutation of survival motor neuron 1 (SMN1) gene. Although survival motor neuron 2 (SMN2) gene is still present in SMA patients, the production of full-length survival motor neuron (SMN) protein is insufficient owing to missing or mutated SMN1. No current disease-modifying therapies can cure SMA. The aim of this study was to explore microRNA (miRNA)-based therapies that may serve as a potential target for therapeutic intervention in delaying SMA progression or as treatment. The study screened for potentially dysregulated miRNAs in SMA fibroblast-derived iPSCs using miRNA microarray. Results from the miRNA microarray were validated using quantitative reverse transcription polymerase chain reaction. Bioinformatics analysis using various databases was performed to predict the potential putative gene targeted by hsa-miR-663a. The findings showed differential expression of hsa-miR-663a in SMA patients in relation to a healthy control. Bioinformatics analysis identified GNG7, IGF2, and TNN genes that were targeted by hsa-miR-663a to be involved in the PI3K-AKT pathway, which may be associated with disease progression in SMA. Thus, this study suggests the potential role of hsa-miR-663a as therapeutic target for the treatment of SMA patients in the near future.
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Cystic fibrosis (CF) was earlier thought to be a disease prevalent in the West among Caucasians. However, quite a number of recent studies have uncovered CF cases outside of this region, and reported hundreds of unique and novel variant forms of CFTR. Here, we discuss the evidence of CF in parts of the world earlier considered to be rare; Africa, and Asia. This review also highlighted the CFTR mutation variations and new mutations discovered in these regions. This discovery implies that the CF data from these regions were earlier underestimated. The inadequate awareness of the disease in these regions might have contributed towards the poor diagnostic facilities, under-diagnosis or/and under-reporting, and the lack of CF associated health policies. Overall, these regions have a high rate of infant, childhood and early adulthood mortality due to CF. Therefore, there is a need for a thorough investigation of CF prevalence and to identify unique and novel variant mutations within these regions in order to formulate intervention plans, create awareness, develop mutation specific screening kits and therapies to keep CF mortality at bay.
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Hand, foot and mouth disease (HFMD) is a childhood disease caused by enterovirus A71 (EV-A71) and coxsackievirus A16 (CV-A16). Capsid loops are important epitopes for EV-A71 and CV-A16. Seven chimeric EV-A71 (ChiE71) involving VP1 BC (45.5% similarity), DE, EF, GH and HI loops, VP2 EF loop and VP3 GH loop (91.3% similarity) were substituted with corresponding CV-A16 loops. Only ChiE71-1-BC, ChiE71-1-EF, ChiE71-1-GH and ChiE71-3-GH were viable. EV-A71 and CV-A16 antiserum neutralized ChiE71-1-BC and ChiE71-1-EF. Mice immunized with inactivated ChiE71 elicited high IgG, IFN-γ, IL-2, IL-4 and IL-10. Neonatal mice receiving passive transfer of WT EV-A71, ChiE71-1-EF and ChiE71-1-BC immune sera had 100%, 80.0% and no survival, respectively, against lethal challenges with EV-A71, suggesting that the substituted CV-A16 loops disrupted EV-A71 immunogenicity. Passive transfer of CV-A16, ChiE71-1-EF and ChiE71-1-BC immune sera provided 40.0%, 20.0% and 42.9% survival, respectively, against CV-A16. One-day-old neonatal mice immunized with WT EV-A71, ChiE71-1-BC, ChiE71-1-EF and CV-A16 achieved 62.5%, 60.0%, 57.1%, and no survival, respectively, after the EV-A71 challenge. Active immunization using CV-A16 provided full protection while WT EV-A71, ChiE71-1-BC and ChiE71-1-EF immunization showed partial cross-protection in CV-A16 lethal challenge with survival rates of 50.0%, 20.0% and 40%, respectively. Disruption of a capsid loop could affect virus immunogenicity, and future vaccine design should include conservation of the enterovirus capsid loops.
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BACKGROUND: In coronavirus disease 2019 (COVID-19) patients, elevated levels of inflammatory cytokines from over stimulation of immune cells have become a concern due to the potential outburst of cytokine storm that damages the tissues and organs, especially the lungs. This leads to the manifestation of COVID-19 symptoms, such as pneumonia, acute respiratory distress syndrome (ARDS), multiple organ failure, and eventually death. Mesenchymal stromal/stem cells (MSCs) are currently one of hopeful approaches in treating COVID-19 considering its anti-inflammatory and immunomodulatory functions. On that account, the number of clinical trials concerning the use of MSCs for COVID-19 has been increasing. However, the number of systematic reviews and meta-analysis that specifically discuss its potential as treatment for the disease is still lacking. Therefore, this review will assess the safety and efficacy of MSC administration in COVID-19 patients. OBJECTIVES: To pool evidence on the safety and efficacy of MSCs in treating COVID-19 by observing MSC-related adverse effects as well as evaluating its effects in reducing inflammatory response and improving pulmonary function. DATA SOURCES AND METHODS: Following literature search across six databases and one trial register, full-text retrieval, and screening against eligibility criteria, only eight studies were included for data extraction. All eight studies evaluated the use of umbilical cord-derived mesenchymal stromal/stem cell (UC-MSC), infused intravenously. Of these eight studies, six studies were included in meta-analysis on the incidence of mortality, adverse events (AEs), and serious adverse events (SAEs), and the levels of C-reactive protein (CRP) and interleukin (IL)-6. Meta-analysis on pulmonary function was not performed due to insufficient data. RESULTS: MSC-treated group showed significantly lower risk of mortality than the control group (p = 0.03). No statistical significance was observed on the incidence of AEs (p = 0.78) and SAEs (p = 0.44), and the levels of CRP (p = 0.06) and IL-6 (p = 0.09). CONCLUSION: MSCs were safe for use, with lower risk of mortality and no association with AEs. Regarding efficacy, descriptive analysis showed indications of improvement on the inflammatory reaction, lung clearance, and oxygenation status despite the lack of statistical significance in meta-analysis of CRP and IL-6. Nevertheless, more studies are needed for affirmation. REGISTRATION: This systematic review and meta-analysis was registered on the PROSPERO database (no. CRD42022307730).
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COVID-19 , Transplante de Células-Tronco Mesenquimais , Células-Tronco Mesenquimais , Humanos , SARS-CoV-2/metabolismo , Interleucina-6/metabolismo , Transplante de Células-Tronco Mesenquimais/efeitos adversos , Citocinas/metabolismo , Células-Tronco Mesenquimais/metabolismoRESUMO
Bladder cancer cells can acquire persistent infection of oncolytic Newcastle disease virus (NDV) but the molecular mechanism(s) remain unelucidated. This poses a major barrier to the effective clinical translation of oncolytic NDV virotherapy of cancers. To improve our understanding of the molecular mechanism(s) associated with the development of NDV persistent infection in bladder cancer, we used mRNA expression profiles of persistently infected bladder cancer cells to construct PPI networks. Based on paths and modules in the PPI network, the bridges were found mainly in the upregulated mRNA-pathways of p53 signalling, ECM-receptor interaction, and TGF-beta signalling and downregulated mRNA-pathways of antigen processing and presentation, protein processing in endoplasmic reticulum, completement and coagulation cascades in persistent TCCSUPPi cells. In persistent EJ28Pi cells, connections were identified mainly through upregulated mRNA-pathways of renal carcinoma, viral carcinogenesis, Ras signalling and cell cycle and the downregulated mRNA-pathways of Wnt signalling, HTLV-I infection and pathways in cancers. These connections were mainly dependent on RPL8-HSPA1A/HSPA4 in TCCSUPPi cells and EP300, PTPN11, RAC1-TP53, SP1, CCND1 and XPO1 in EJ28Pi cells. Oncomine validation showed that the top hub genes identified in the networks that include RPL8, THBS1, F2 from TCCSUPPi and TP53 and RAC1 from EJ28Pi are involved in the development and progression of bladder cancer. Protein-drug interaction networks identified several putative drug targets that could be used to disrupt the linkages between the modules and prevent bladder cancer cells from acquiring NDV persistent infection. This novel PPI network analysis of differentially expressed mRNAs of NDV persistently infected bladder cancer cell lines provide an insight into the molecular mechanisms of NDV persistency of infection in bladder cancers and the future screening of drugs that can be used together with NDV to enhance its oncolytic efficacy.
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Doença de Newcastle , Terapia Viral Oncolítica , Vírus Oncolíticos , Neoplasias da Bexiga Urinária , Animais , Humanos , Vírus da Doença de Newcastle/genética , Transcriptoma , Linhagem Celular Tumoral , Infecção Persistente , Vírus Oncolíticos/genética , Neoplasias da Bexiga Urinária/genética , RNA Mensageiro/genéticaRESUMO
Plasmid DNA is one of the indispensable components in molecular biology research and a potential biomaterial for gene therapy and DNA vaccination. Both quality and quantity of extracted plasmid DNA are of the great interests in cloning and subsequent expression of genes in vitro and in vivo for basic research and therapeutic interventions. Bacteria with extremely short generation times are the valuable source of plasmid DNA that can be isolated through a number of existing techniques. However, the current methods have some limitations in isolating high quality plasmid DNA since the multimeric plasmid which is believed to be more efficiently transcribed by RNA polymerase than the monomeric form, is almost lost during the extraction process. Recently, we developed a rapid isolation technique for multimeric plasmid based on generation of a 'protein aggregate' using a zwitterionic detergent and alkali. Here we have investigated the roles of different parameters in the whole extraction process to optimise the production of high quality multimeric plasmid DNA. Moreover, we have showed the advantageous effects of nanoparticles to effectively sediment the 'protein aggregate' for smooth elution of multimeric plasmid DNA from it. Finally, quality assessment study has revealed that the isolated multimeric DNA is at least 10 times more transcriptionally active than the monomeric form isolated by the commercially available Qiaget kit.
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Apatitas/química , DNA Bacteriano/isolamento & purificação , Plasmídeos/isolamento & purificação , Compostos de Amônio Quaternário/química , Soluções Tampão , DNA Bacteriano/genética , Escherichia coli/genética , Escherichia coli/crescimento & desenvolvimento , Células HeLa , Humanos , Nanopartículas/química , Plasmídeos/química , Plasmídeos/genética , Transfecção , Transformação BacterianaRESUMO
The underlying mechanisms by which Newcastle disease virus (NDV) kills cancer cells are still unclear. Recent discoveries have shown that many viruses contain Bcl-2 homology-like domains which enabled their interaction with Bcl-2 family members, and thereby accounting for their virulence and pathogenicity. Alignment of the protein sequences of Malaysian strain of NDV, known as AF2240, with those from members of the human Bcl-2 family showed many similar regions; most notably we found that its matrix (AF2240-M) protein, large (AF2240-L) protein and fusion (AF2240-F) protein all contain BH3-like regions. In addition, there are BH1-like domains in these proteins, where AF2240-F and Mcl-1 share 55% identity within this region. To further investigate our hypothesis that the presence of the BH3-like domains in these proteins may convey cytotoxicity, AF2240-M and AF2240-F genes were cloned into pFLAG and pEGFP.N2 vectors and transfected into HeLa cells. The expression of these constructs promoted cell death. As shown by flow cytometry, AF2240-M protein with deleted BH3-like region showed five-fold decrease in apoptosis. Moreover, the construct containing the N-terminal of AF2240-M showed nearly the same cell death rate as to that of the full-length protein, strongly suggesting that the BH3-like domain within this protein participates in promoting cell death. Moreover, AF2240-M transfection promoted Bax redistribution to mitochondria. Therefore, to determine whether there is any direct interaction between NDV viral proteins with some members of the Bcl-2 family, various constructs were co-transfected into HeLa cells. Co-immunoprecipitation trials showed that the AF2240-M indeed directly interacted with Bax protein via its BH3-domain, as the mutant proteins failed to interact with Bax. AF2240-F failed to interact with any of the tested proteins, although Bcl-XL slowed down the rate of cell death caused by this construct by nearly five-fold. In a parallel experiment, the level of expression of endogenous Bax and Bcl-2 after infection of HeLa cells with NDV was assessed by qRT-PCR, but no statistically significant change was observed. Consequently, the Bax/Bcl-2 ratio at the mRNA level did not alter. Overall, our study has shed additional light into the mechanisms by which NDV induces apoptosis.
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Interações Hospedeiro-Patógeno , Vírus da Doença de Newcastle/patogenicidade , Domínios e Motivos de Interação entre Proteínas , Proteínas da Matriz Viral/metabolismo , Proteína X Associada a bcl-2/metabolismo , Apoptose , Genes Reporter , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Imunoprecipitação , Ligação Proteica , TransfecçãoRESUMO
Direct reprogramming of somatic cells into induced pluripotent stem (iPS) cells has emerged as an invaluable method for generating patient-specific stem cells of any lineage without the use of embryonic materials. Following the first reported generation of iPS cells from murine fibroblasts using retroviral transduction of a defined set of transcription factors, various new strategies have been developed to improve and refine the reprogramming technology. Recent developments provide optimism that the generation of safe iPS cells without any genomic modification could be derived in the near future for the use in clinical settings. This review summarizes current and evolving strategies in the generation of iPS cells, including types of somatic cells for reprogramming, variations of reprogramming genes, reprogramming methods, and how the advancement iPS cells technology can lead to the future success of reproductive medicine.
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Rearranjo Gênico , Células-Tronco Pluripotentes Induzidas/citologia , Transfecção/métodos , Animais , Técnicas de Cultura de Células , Diferenciação Celular , Clonagem Molecular , Fibroblastos/citologia , Técnicas de Transferência de Genes , Vetores Genéticos/genética , Humanos , Camundongos , MicroRNAs/genética , MicroRNAs/metabolismo , Plasmídeos , Técnicas Reprodutivas , Retroviridae/genética , Fatores de Transcrição/genética , Transdução Genética/métodosRESUMO
Spinal muscular atrophy (SMA) is a neurodegenerative disease caused by low levels of full-length survival motor neuron (SMN) protein due to the loss of the survival motor neuron 1 (SMN1) gene and inefficient splicing of the survival motor neuron 2 (SMN2) gene, which mostly affects alpha motor neurons of the lower spinal cord. Despite the U.S. Food and Drug Administration (FDA) approved SMN-dependent therapies including Nusinersen, Zolgensma® and Evrysdi™, SMA is still a devastating disease as these existing expensive drugs may not be sufficient and thus, remains a need for additional therapies. The involvement of microRNAs (miRNAs) in SMA is expanding because miRNAs are important mediators of gene expression as each miRNA could target a number of genes. Hence, miRNA-based therapy could be utilized in treating this genetic disorder. However, the delivery of miRNAs into the target cells remains an obstacle in SMA, as there is no effective delivery system to date. This review highlights the potential strategies for intracellular miRNA delivery into target cells and current challenges in miRNA delivery. Furthermore, we provide the future prospects of miRNA-based therapeutic strategies in SMA.
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MicroRNAs , Atrofia Muscular Espinal , Doenças Neurodegenerativas , Animais , Modelos Animais de Doenças , Humanos , MicroRNAs/genética , Neurônios Motores/patologia , Atrofia Muscular/patologia , Atrofia Muscular Espinal/genética , Atrofia Muscular Espinal/patologia , Atrofia Muscular Espinal/terapia , Doenças Neurodegenerativas/patologia , Proteína 1 de Sobrevivência do Neurônio MotorRESUMO
Pre-clinical studies have demonstrated the efficacy of mesenchymal stem cells (MSCs) expressing tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL) or MSC-TRAIL against several tumors. However, due to the existence of cancer stem cells (CSCs), some tumors, including non-small cell lung cancer (NSCLC), exhibit TRAIL resistance. This study was designed to evaluate the capacity of using first-line chemotherapies including cisplatin, 5-fluorouracil (5-FU) and vinorelbine to act as a chemo-sensitizer on CD133+ (prominin-1 positive) CSCs derived from NSCLC cell lines (A549, H460 and H2170) for the purpose of MSC-TRAIL-induced inhibition. We showed that MSC-TRAIL was resistant to all three chemotherapies compared to the NSCLC cell lines, suggesting that the chemotherapies had little effect on MSC-TRAIL viability. Pre-treatment using either cisplatin or 5-FU, but not with vinorelbine, was able to increase the efficacy of MSC-TRAIL to kill the TRAIL-resistant A549-derived CSCs. The study also demonstrated that both 5-FU and vinorelbine were an effective chemo-sensitizer, used to increase the anti-tumor effect of MSC-TRAIL against H460- and H2170-derived CSCs. Furthermore, pre-treatment using cisplatin was noted to enhance the effect of MSC-TRAIL in H460-derived CSCs; however, this effect was not detected in the H2170-derived CSCs. These findings suggest that a pre-treatment using certain chemotherapies in NSCLC could enhance the anti-tumor effect of MSC-TRAIL to target the CSCs, and therefore the combination of chemotherapies and MSC-TRAIL may serve as a novel approach for the treatment of NSCLC.
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The development of a T7 RNA polymerase (T7 RNAP) expressing cell line i.e. BSR T7/5 cells marks an improvement of reverse genetics for the recovery of recombinant Newcastle disease virus (rNDV). BSR T7/5 is developed by transient transfection of plasmid encoding T7 RNAP gene for rNDV rescue. However, the gene expression decreases gradually over multiple passages and eventually hinders the rescue of rNDV. To address this issue, lentiviral vector was used to develop T7 RNAP-expressing HEK293-TA (HEK293-TA-Lv-T7) and SW620 (SW620-Lv-T7) cell lines, evidenced by the expression of T7 RNAP after subsequent 20 passages. rNDV was rescued successfully using HEK293-TA-Lv-T7 clones (R1D3, R1D8, R5B9) and SW620-Lv-T7 clones (R1C11, R3C5) by reverse transfection, yielding comparable virus rescue efficiency and virus titres to that of BSR T7/5. This study provides new tools for rNDV rescue and insights into cell line development and virology by reverse genetics.
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Lentivirus , Vírus da Doença de Newcastle , Animais , RNA Polimerases Dirigidas por DNA/genética , Vetores Genéticos , Células HEK293 , Humanos , Lentivirus/genética , Vírus da Doença de Newcastle/genética , Plasmídeos , Transfecção , Proteínas ViraisRESUMO
A novel cationic polymer, dextran-spermine (D-SPM), has been found to mediate gene expression in a wide variety of cell lines and in vivo through systemic delivery. Here, we extended the observations by determining the optimal conditions for gene expression of D-SPM/plasmid DNA (D-SPM/pDNA) in cell lines and in the lungs of BALB/c mice via instillation delivery. In vitro studies showed that D-SPM could partially protect pDNA from degradation by nuclease and exhibited optimal gene transfer efficiency at D-SPM to pDNA weight-mixing ratio of 12. In the lungs of mice, the levels of gene expression generated by D-SPM/pDNA are highly dependent on the weight-mixing ratio of D-SPM to pDNA, amount of pDNA in the complex, and the assay time postdelivery. Readministration of the complex at day 1 following the first dosing showed no significant effect on the retention and duration of gene expression. The study also showed that there was a clear trend of increasing size of the complexes as the amount of pDNA was increased, where the sizes of the D-SPM/pDNA complexes were within the nanometer range.
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Dextranos/química , Técnicas de Transferência de Genes , Pulmão/metabolismo , Nanopartículas , Plasmídeos/genética , Espermina/química , Animais , Células COS , Chlorocebus aethiops , DNA , Desoxirribonuclease I , Eletroforese em Gel de Ágar , Citometria de Fluxo , Expressão Gênica , Genes Reporter , Luciferases , Substâncias Macromoleculares , Camundongos , Camundongos Endogâmicos BALB C , Microscopia de Fluorescência , Tamanho da PartículaRESUMO
The generation of induced pluripotent stem cells (iPSCs) from differentiated mature cells is one of the most promising technologies in the field of regenerative medicine. The ability to generate patient-specific iPSCs offers an invaluable reservoir of pluripotent cells, which could be genetically engineered and differentiated into target cells to treat various genetic and degenerative diseases once transplanted, hence counteracting the risk of graft versus host disease. In this context, we review the scientific research streams that lead to the emergence of iPSCs, the roles of reprogramming factors in reprogramming to pluripotency, and the reprogramming strategies. As iPSCs serve tremendous correction potentials for various diseases, we highlight the successes and challenges of iPSCs in cell replacement therapy and the synergy of iPSCs and clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 gene editing tools in therapeutics research.
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CpG-free pDNA was reported to facilitate sustained transgene expression with minimal inflammation in vivo as compared to CpG-containing pDNA. However, the expression potential and impact of CpG-free pDNA in in vitro model have never been described. Hence, in this study, we analyzed the transgene expression profiles of CpG-free pDNA in vitro to determine the influence of CpG depletion from the transgene. We found that in contrast to the published in vivo studies, CpG-free pDNA expressed a significantly lower level of luciferase than CpG-rich pDNA in several human cell lines. By comparing novel CpG-free pDNA carrying CpG-free GFP (pZGFP: 0 CpG) to CpG-rich GFP (pRGFP: 60 CpGs), we further showed that the discrepancy was not influenced by external factors such as gene transfer agent, cell species, cell type, and cytotoxicity. Moreover, pZGFP exhibited reduced expression despite having equal gene dosage as pRGFP. Analysis of mRNA distribution revealed that the mRNA export of pZGFP and pRGFP was similar; however, the steady state mRNA level of pZGFP was significantly lower. Upon further investigation, we found that the CpG-free transgene in non-integrating CpG-free pDNA backbone acquired increased nucleosome enrichment as compared with CpG-rich transgene, which may explain the observed reduced level of steady state mRNA. Our findings suggest that nucleosome enrichment could regulate non-integrating CpG-free pDNA expression and has implications on pDNA design.
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Ilhas de CpG , Nucleossomos/genética , Plasmídeos/genética , Transgenes , Animais , Linhagem Celular , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Células HEK293 , Humanos , Luciferases/genética , Células MCF-7 , Camundongos , Células NIH 3T3 , Especificidade da Espécie , TransfecçãoRESUMO
Short-lived therapeutic gene expression in mammalian cells by DNA methylation is one of the major challenges in gene therapy. In this study, we assessed the implication of DNA methylation on the duration of GFP expression in mouse embryonic stem (ES) and mouse induced pluripotent stem (iPS) cells. The cells were transduced with lentivirus (LV) carrying green fluorescent protein (GFP) driven by either human elongation factor (EF1α) or cytomegalovirus (CMV) promoter. Transduced iPS cells exhibited higher percentage of GFP+ cells with persistent mean fluorescent intensity than transduced ES cells. Analysis on the integrated copy of transgene in the population of the transduced cells demonstrated similar copy number. However, significant increase in GFP intensity following 5-azaC treatment was observed in transduced ES cells only, suggesting the influence of DNA methylation in transgene silencing. Subsequent DNA methylation analysis showed that the promoter and the GFP region of the provirus in iPS cells had negligible methylation profile compared to transduced ES cells. Interestingly, sustained transgene expression was observed upon directed differentiation of transduced iPS cells towards CD34+ CD45+ cells. Hence, this study has shown that favourable transgene activity from lentiviral transduced iPS cells was due to the lack of methylation at the proviral regions.
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Metilação de DNA/genética , Células-Tronco Embrionárias/metabolismo , Proteínas de Fluorescência Verde/genética , Células-Tronco Pluripotentes Induzidas/metabolismo , Animais , Azacitidina/farmacologia , Linhagem Celular , Citomegalovirus/genética , Fator de Iniciação 1 em Eucariotos/genética , Regulação da Expressão Gênica/genética , Células HEK293 , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Regiões Promotoras Genéticas/genética , Transdução GenéticaRESUMO
DNA methylation and histone modifications are vital in maintaining genomic stability and modulating cellular functions in mammalian cells. These two epigenetic modifications are the most common gene regulatory systems known to spatially control gene expression. Transgene silencing by these two mechanisms is a major challenge to achieving effective gene therapy for many genetic conditions. The implications of transgene silencing caused by epigenetic modifications have been extensively studied and reported in numerous gene delivery studies. This review highlights instances of transgene silencing by DNA methylation and histone modification with specific focus on the role of these two epigenetic effects on the repression of transgene expression in mammalian cells from integrative and non-integrative based gene delivery systems in the context of gene therapy. It also discusses the prospects of achieving an effective and sustained transgene expression for future gene therapy applications.