17 research outputs found
Production of lentivirus carrying green fluorescent protein with different promoters for in vitro gene transfer
Many diseases are potential targets for gene therapy using either non-viral or viral vectors. Unlike non-viral methods, viral vectors, such as lentiviruses, have the ability to integrate into the host chromosome, which can lead to long-term transgene expression. Lentiviruses have advantages over other types of viruses due to their capacity to transduce non-dividing cells. An optimized generation of lentiviruses carrying green fluorescent protein (GFP) reporter gene driven by either UbC (LV/UbC/GFP) or CMV (LV/CMV/GFP) promoter is described in this paper. The lentiviruses were produced by co-transfecting lentiviral expression constructs and packaging mix into 293FT lentivirus producer cell lines. Lipofectamine was highly efficient in transfecting the cells compared to Transfast and Polyethyleneimine (PEI). Following cell transfection, syncytia were clearly visible at day 2. Lentiviruses were harvested at days 1, 2 and 3 post-transfection. The highest transduction efficiency was read from LV/CMV/GFP harvested at day 2 post-transfection and LV/UbC/GFP harvested at day 3 post-transfection. Finally, the GFP expression in COS-7 cells was determined at day 2 and day 14 post-transduction for transient and stable GFP expression. It was found that the GFP expression declined overtime. However, the transduction efficiency and duration of the transgene expression in COS-7 cells transduced with LV/CMV/GFP were higher compared to LV/UbC/GFP. In conclusion, we have successfully produced lentiviruses carrying GFP with different promoters and shown that the viruses were able to infect COS-7 cells at different efficiencies. Meanwhile, the generation of the active lentiviruses will allow us to the subsequent analysis of the effect of regulatory elements in future study
Production of lentiviral vector with polycistronic transcripts for reprogramming of mouse fibroblast cells
iPS cells were originally generated using monocistronic retroviral vectors carrying the Yamanaka factors 'OSKM'. The development of a polycistronic viral vector with OSKM linked by 2A peptides has simplified reprogramming procedure and reduced the risk of multiple proviral integrations and insertional mutagenesis. In this study, we demonstrated the production of the polycistronic lentiviral vector encoding OSKM in a single cassette without a reporter gene or drug-based selection system. Syncytia formations were clearly seen following the co-transfection of a lentiviral plasmid construct with the structural and packaging plasmids. The virion was collected at 48 hours post-transfection. Afterwards, the viral titers were measured by the expression of Sox2 protein from transduced HT1080 cells. Subsequently, Oct4 expression was successfully detected in mouse fibroblasts in the range of 5, 10 and 20 MOIs with expression of 90.7%, 97.5% and 98%, respectively. The results obtained from this study could be used as a model for the production of OSKM lentiviral vector for newcomers to cellular reprogramming research
Induced pluripotent stem cells: reprogramming platforms and applications in cell replacement therapy
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
Lack of methylation on transgene leads to high level and persistent transgene expression in induced pluripotent stem cells
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
Zebularine and Trichostatin A sensitised human breast adenocarcinoma cells towards tumour necrosis factor-related apoptosis inducing ligand (TRAIL)-induced apoptosis
Tumour necrosis factor-related apoptosis-inducing ligand (TRAIL) is a promising cancer therapeutic agent due to its selective killing on cancer cells while sparing the normal cells. Nevertheless, breast adenocarcinoma cells can develop TRAIL resistance. Therefore, this project investigated the anti-cancer effects of the combination of epigenetic drugs zebularine and trichostatin A (ZT) with TRAIL (TZT) on the human breast adenocarcinoma cells. This treatment regimen was compared with the natural anti-cancer compound curcumin (Cur) and standard chemotherapeutic drug doxorubicin (Dox). As compared to TRAIL treatment, TZT treatment hampered the cell viability of human breast adenocarcinoma cells MDA-MB-231 significantly but not MCF-7 and immortalized non-cancerous human breast epithelial cells MCF10A. Unlike TZT, Cur and Dox treatments reduced cell viability in both human breast adenocarcinoma and epithelial cells significantly. Nevertheless, there were no changes in cell cycle in both TRAIL and TZT treatments in breast adenocarcinoma and normal epithelial cells. Intriguingly, Cur and Dox treatment generally induced G2/M arrest in MDA-MB-231, MCF-7 and MCF10A but Cur induced S phase arrest in MCF10A. The features of apoptosis such as morphological changes, apoptotic activity and the expression of cleaved poly (ADP) ribose polymerase (PARP) protein were more prominent in TRAIL and TZT-treated MDA-MB-231 as compared to MCF10A at 24 h post-treatment. Compared to TZT treatment, Cur and Dox treatments exhibited lesser apoptotic features in MDA-MB-231. Collectively, the sensitization using Zeb and TSA to augment TRAIL-induced apoptosis might be an alternative therapy towards human breast adenocarcinoma cells, without harming the normal human breast epithelial cells
Mindfulness-based supportive therapy on reducing suffering in patients with advanced cancer: randomised controlled trial
Objectives Suffering is common among
patients with advanced cancer. The practice of
mindfulness during patient care can potentially
reduce suffering. We aimed to examine the
efficacy of mindfulness-based supportive therapy
(MBST) on reducing suffering in patients with
advanced cancer.
Methods We conducted a parallel-group,
single-blinded, randomised controlled trial at the
University of Malaya Medical Centre, Malaysia.
Seventy-three patients with advanced cancer
with an overall suffering score ≥4/10 based on
the Suffering Pictogram were recruited and
randomly assigned into either the MBST group
(n=34) or the control group (n=39).
Results There was a statistically significant
reduction in the overall suffering score in the
MBST group compared with the control group
(U=432.5, median1
=−2.0, median2
=−1.0,
z=−2.645, p=0.008). There was also significant
improvement in the total Hospital Anxiety and
Depression Scale score (U=483.5, median1
=−4.0,
median2
=−3.0, z=−1.994, p=0.046), and the
total Functional Assessment of Chronic Illness
Therapy-Spiritual Well-Being score (U=252.0,
median1
=+14.5, median2
=+5.0, z=−4.549,
p=0.000) in the MBST group compared with the
control group.
Conclusions The results provided evidence that
the practice of MBST during patient care could
promote positive psychosocial outcome
DNA Methylation and Histone Modifications Are the Molecular Lock in Lentivirally Transduced Hematopoietic Progenitor Cells
Stable introduction of a functional gene in hematopoietic progenitor cells (HPCs) has appeared to be an alternative approach to correct genetically linked blood diseases. However, it is still unclear whether lentiviral vector (LV) is subjected to gene silencing in HPCs. Here, we show that LV carrying green fluorescent protein (GFP) reporter gene driven by cytomegalovirus (CMV) promoter was subjected to transgene silencing after transduction into HPCs. This phenomenon was not due to the deletion of proviral copy number. Study using DNA demethylating agent and histone deacetylase (HDAC) inhibitor showed that the drugs could either prevent or reverse the silencing effect. Using sodium bisulfite sequencing and chromatin immunoprecipitation (ChIP) assay, we demonstrated that DNA methylation occurred soon after LV transduction. At the highest level of gene expression, CMV promoter was acetylated and was in a euchromatin state, while GFP reporter gene was acetylated but was strangely in a heterochromatin state. When the expression declined, CMV promoter underwent transition from acetylated and euchromatic state to a heterochromatic state, while the GFP reporter gene was in deacetylated and heterochromatic state. With these, we verify that DNA methylation and dynamic histone modifications lead to transgene silencing in HPCs transduced with LV
Transgene expression from CpG-reduced lentiviral gene delivery vectors in vitro
Current viral gene delivery vectors for gene therapy are inefficient due to short-lived transgene expression attributed to the cytosine-phosphate-guanine (CpG) motifs in the transgene. Here we assessed the effects of CpG motif reduction in lentiviral (LV) gene delivery context on the level and duration of reporter gene expression in Chinese Hamster Ovary (CHO) cells, Human Immortalized Myelogenous Leukemia (K562) cells and hematopoietic stem cells (HSCs). The cells were transduced with LV carrying Zero-CpG green fluorescent protein (ZGFP) reporter gene, LV/CMV/ZGFP. The GFP expression was compared to its non CpG-depleted GFP reporter gene LV (LV/CMV/GFP) counterpart. The LV/CMV/ZGFP exhibited prolonged transgene expression in CHO cells and HSCs up to 10 days and 14 days, in the respective cells. This effect was not seen in the transduced K562 cells, which may be due to the DNA hypomethylation status of the cancer cell line. Transgene copy number analysis verified that the GFP expression was not from pseudo-transduction and the transgene remained in the genome of the cells throughout the period of the study. The modest positive effects from the LV/CMV/ZGFP suggest that the reduction of CpG in the LV construct was not substantial to generate higher and more prolonged transgene expression
The Role of Ras-Associated Protein 1 (Rap1) in Cancer: Bad Actor or Good Player?
Metastasis is known as the most life-threatening event in cancer patients. In principle, the immune system can prevent tumor development. However, dysfunctional T cells may fail to eliminate the tumor cells effectively and provide additional survival advantages for tumor proliferation and metastasis. Constitutive activation of Ras-associated protein1 (Rap1) has not only led to T cell anergy, but also inhibited autophagy and supported cancer progression through various oncogenic events. Inhibition of Rap1 activity with its negative regulator, Rap1GAP, impairs tumor progression. However, active Rap1 reduces tumor invasion in some cancers, indicating that the pleiotropic effects of Rap1 signaling in cancers could be cancer-specific. All in all, targeting Rap1 signaling and its regulators could potentially control carcinogenesis, metastasis, chemoresistance and immune evasion. Rap1GAP could be a promising therapeutic target in combating cancer
Curcumin Nanoformulations for Colorectal Cancer: A Review
Colorectal cancer (CRC) is the third most prevalent form of cancer, after lung cancer and breast cancer, with the second highest death incidence. Over the years, natural compounds have been explored as an alternative to conventional cancer therapies such as surgery, radiotherapy, and chemotherapy. Curcumin, an active constituent of turmeric has been associated with various health benefits. It has gained much attention as an anticancer agent due to its ability to regulate multiple cell signaling pathways, including NF-κB, STAT3, activated protein-1 (AP-1), epidermal growth response-1 (Egr-1), and p53, which are crucial in cancer development and progression. Nevertheless, the clinical application of curcumin is greatly restricted because of its low water solubility, poor oral absorption, and rapid metabolism. These issues have led to the development of curcumin nanoformulations to overcome the limitations of the compound. Nanotechnology-based delivery systems have been widely used in improving the delivery of poorly-water soluble drugs. Besides, these systems also come with the added benefits of possible cellular targeting and improvement in cellular uptake. An ideal improved formulation should display a greater anticancer activity compared to free curcumin, and at the same time be non-toxic to the normal cells. In this review, we focus on the design and development of various nanoformulations to deliver curcumin for use in CRC such as liposomes, micelles, polymer nanoparticles, nanogels, cyclodextrin complexes, solid lipid nanoparticles (SLN), phytosomes, and gold nanoparticles. We also discuss the current pre-clinical and clinical evidences of curcumin nanoformulations in CRC therapy, analyse the research gap, and address the future direction of this research area. © 2019 Wong, Ngai, Chan, Lee, Goh and Chuah