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.

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.

Silencing of transgene expression in mammalian cells by DNA methylation and histone modifications in gene therapy perspective.

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.

Gene delivery potential of biofunctional carbonate apatite nanoparticles in lungs.

Existing nonviral gene delivery systems to lungs are inefficient and associated with dose limiting toxicity in mammalian cells. Therefore, carbonate apatite (CO3Ap) nanoparticles were examined as an alternative strategy for effective gene delivery to the lungs. This study aimed to (1) assess the gene delivery efficiency of CO3Ap in vitro and in mouse lungs, (2) evaluate the cytotoxicity effect of CO3Ap/pDNA in vitro, and (3) characterize the CO3Ap/pDNA complex formulations. A significantly high level of reporter gene expression was detected from the lung cell line transfected with CO3Ap/pDNA complex prepared in both serum and serum-free medium. Cytotoxicity analysis revealed that the percentage of the viable cells treated with CO3Ap to be almost similar to the untreated cells. Characterization analyses showed that the CO3Ap/pDNA complexes are in a nanometer range with aggregated spherical structures and tended to be more negatively charged. In the lung of mice, highest level of transgene expression was observed when CO3Ap (8 µL) was complexed with 40 µg of pDNA at day 1 after administration. Although massive reduction of gene expression was seen beyond day 1 post administration, the level of expression remained significant throughout the study period.

Comparison of Ketorolac Tromethamine and Prednisolone Acetate in Preventing Surgically Induced Miosis during Cataract Surgery.

OBJECTIVES: The aim of this study was to compare the efficacy and safety of topical prednisolone acetate 1% and topical ketorolac tromethamine 0.5% in the maintenance of pupillary mydriasis during cataract surgery. METHODS: Fifty patients were enrolled in this prospective, partially masked and randomised study. They were assigned to receive topical treatment with either prednisolone acetate (n = 25) or ketorolac tromethamine (n = 25), starting 24 hours before cataract extraction (either routine extracapsular cataract extraction or phacoemulsification). One drop of the study medication was instilled every 6 hours for a total of 4 drops. No epinephrine was used in the intraoperative irrigation solution. Pupil diameter was measured three different times during surgery. To ensure participant safety, biomicroscopy, ophthalmoscopy, intraocular pressure, adverse events and visual acuity were also monitored. RESULTS: The mean pupil diameter change from the time of the pre-incision until after cortical irrigation and aspiration and lens implantation was significantly less with ketorolac than with prednisolone (P = 0.003). Consequently, mean pupil diameter after cortical irrigation and aspiration and lens implantation was significantly greater with ketorolac than with prednisolone (P <0.0001). No significant differences between groups were observed in the pupil diameter before the first incision (P = 0.244), nor after administration of a miotic agent (P = 0.505). Safety variables were comparable and no drug-related adverse events were reported. CONCLUSION: Ketorolac tromethamine 0.5% and prednisolone acetate 1% solutions were equally well tolerated without related adverse events, but ketorolac was better in preventing surgically induced miosis.

Prevalence and risk factors for pulmonary artery systolic hypertension among sickle cell disease patients in Nigeria.

Pulmonary artery systolic hypertension is common and associated with increased mortality among adult sickle cell disease (SCD) patients in the United States. Although the prevalence of SCD is highest in sub-Saharan Africa, the frequency of pulmonary artery systolic hypertension and the risk factors for the development of pulmonary hypertension have not been reported from Africa. We studied 208 hydroxyurea naïve Nigerian SCD patients at steady state and 94 healthy controls. Pulmonary artery systolic hypertension was defined prospectively as tricuspid regurgitant jet velocity > or =2.5 m/sec. Results were compared with a previously published US prospective SCD cohort. Only 7% of Nigerians compared with 46% of US adults with SCD were >35 years. Tricuspid regurgitant jet velocity was > or =2.5 m/sec in 25% of Nigerian SCD patients. Higher jet velocity was associated with greater serum globulin (P = 0.002), blood urea nitrogen (P = 0.019) and lactate dehydrogenase concentrations (P = 0.026) and with inability to walk >300 m in 6 min (P = 0.042). Compared with the US cohort, Nigerian patients had more hemolysis as indicated by lower hemoglobin and higher lactate dehydrogenase concentrations (P < or = 0.003). Pulmonary hypertension is common among Nigerian SCD patients. The public health implication of this finding is significant considering the potential number of individuals at risk for this complication. Better understanding of the long term outcome of pulmonary hypertension and causes of death in SCD and the institution of preventive measures are major public health challenges for Africa. The inclusion of African sites in sickle cell pulmonary hypertension clinical trials should be encouraged.