Safety profile of dextran-spermine gene delivery vector in mouse lungs

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-α, IL-12, IFN-γ 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

Tumour suppressive effects of WEE1 gene silencing in breast cancer cells.

Background: WEE1 is a G2/M checkpoint regulator protein. Various studies have indicated that WEE1 could be a good target for cancer therapy. The main aim of this study was to asssess the tumor suppressive potential of WEE1 silencing in two different breast cancer cell lines, MCF7 which carries the wild-type p53 and MDA-MB468 which contains a mutant type. Materials and Methods: After WEE1 knockdown with specific shRNAs downstream effects on cell viability and cell cycle progression were determined using MTT and flow cytometry analyses, respectively. Real-time PCR and Western blotting were conducted to assess the effect of WEE1 inhibition on the expression of apoptotic (p53) and anti-apoptotic (Bcl2) factors and also a growth marker (VEGF). Results: The results showed that WEE1 inhibition could cause a significant decrease in the viability of both MCF7 and MDA-MB-468 breast cancer cell lines by more than 50%. Interestingly, DNA content assays showed a significant increase in apoptotic cells following WEE1 silencing. WEE1 inhibition also induced upregulation of the apoptotic marker, p53, in breast cancer cells. A significant decrease in the expression of VEGF and Bcl-2 was observed following WEE1 inhibition in both cell lines. Conclusions: In concordance with previous studies, our data showed that WEE1 inhibition could induce G2 arrest abrogation and consequent cell death in breast cancer cells. Moreover, in this study, the observed interactions between the pro- and anti-apoptotic proteins and decrease in the angiogenesis marker expression confirm the susceptibility to apoptosis and validate the tumor suppressive effect of WEE1 inhibition in breast cancer cells. Interestingly, the levels of the sensitivity to WEE1 silencing in breast cancer cells, MCF7 and MDA-MB468, seem to be in concordance with the level of p53 expression

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

Generation of induced pluripotent stem cells by a polycistronic lentiviral vector in feeder- and serum- free defined culture

Induced pluripotent stem cells (iPSCs) have great potentials for regenerative medicine. However, serious concerns such as the use of the viral-mediated reprogramming strategies and exposure of iPSCs to animal products from feeder cells and serum-containing medium have restricted the application of iPSCs in the clinics. Therefore, the generation of iPSCs with minimal viral integrations and in non-animal sourced and serum-free medium is necessary. In this report, a polycistronic lentiviral vector carrying Yamanaka's factors was used to reprogram mouse fibroblasts into iPSCs in feeder- and xeno-free culture environment. The generated iPSCs exhibited morphology and self-renewal properties of embryonic stem cells (ESCs), expression of specific pluripotent markers, and potentials to differentiate into the three-major distinct specialized germ layers in vitro. The iPSCs were also shown to have the potential to differentiate into neural precursor and neurons in culture, with greater than 95% expression of nestin, Pax6 and βIII-tubulin. This body of work describes an alternative method of generating iPSCs by using polycistronic lentiviral vector that may minimize the risks associated with viral vector-mediated reprogramming and animal derived products in the culture media

The potential role of miRNA therapies in spinal muscle atrophy

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

Spatiotemporal expression profiling and molecular characterisation of miR-344b and miR-344c in the developing mouse brain

MicroRNAs are small non-coding RNAs of about 22 nucleotides that regulate gene expression through inhibition or repression processes during post-transcriptional or translational stages. Studies have shown that miRNAs play a crucial role in spatiotemporal regulation of the brain development. A recent study had shown that miR-344 is expressed in a developing mouse brain. In this study, we focused to characterise the spatiotemporal expression of miR-344b and miR-344c during the development of mouse brain. Out in situ hybridisation studies have shown that both miR-344b and miR-344c were strongly expressed in the germinal layer during the early stages of mouse brain development. Postnatally, expression of miR-344b was not detectable in P1 and adult brains. In contrast, miR-344c was expressed globally in P1 brain and was expressed exclusively in the olfactory bulb and granular cell layer of the cerebellum in the adult mouse brain. A subsequent stemloop RT-qPCR analysis showed that expression of the miR-344b and miR-344c was increased from E11.5 and peaked at E15.5. Postnatally, expression level of the miR-344b was reduced while miR-344c continued to express until adulthood. We further investigated the expression of miR-344b and miR-344c in adult mouse multiple organs and the pancreas showed the highest expression for both miRNAs. Subsequent bioinformatics analysis predicted that miR-344b and miR-344c were found to target a total of 1540 and 863 downstream target genes respectively. Genes associated with transcription regulation and nervous system development were subjected to further screening. We found that Olig2 and Otx2 were predicted as the potential downstream target gene for miR-344b and miR-344c respectively. However, luciferase protein suppression assay showed that the expression of Olig2 and Otx2 were not suppressed by overexpression of miR-344b and miR-344c. In conclusion, miR-344b and miR-344c were expressed in the developing mouse brain and may play a role during early mouse brain development although not directly targeting Olig2 and Otx2

Mucosal genetic immunization through microsphere-based oral carriers.

Polymeric carriers in the form of cellulose acetate phthalate (CAP) and alginate (ALG) microspheres were used for encapsulation of plasmid DNA for oral mucosal immunization. Access into the intestinal mucosa by pVAXI eukaryotic expression plasmid vectors carrying gene-coding sequences, either for the cholera enterotoxin B subunit (cfxB) immunostimulatory antigen or the green fluorescent protein (GFP), delivered from both types of microsphere carriers were examined in orally immunized BALB/c mice. Demonstration of transgene protein expression and IgA antibody responses at local mucosal sites suggest immunological response to a potential oral DNA vaccine formulated within the microsphere carriers

Cellular uptake and anticancer effects of mucoadhesive curcumin-containing chitosan nanoparticles

Curcumin, which is derived from turmeric has gained much attention in recent years for its anticancer activities against various cancers. However, due to its poor absorption, rapid metabolism and elimination, curcumin has a very low oral bioavailability. Therefore, we have formulated mucoadhesive nanoparticles to deliver curcumin to the colon, such that prolonged contact between the nanoparticles and the colon leads to a sustained level of curcumin in the colon, improving the anticancer effect of curcumin on colorectal cancer. The current work entails the ex vivo mucoadhesion study of the formulated nanoparticles and the in vitro effect of mucoadhesive interaction between the nanoparticles and colorectal cancer cells. The ex vivo study showed that curcumin-containing chitosan nanoparticles (CUR-CS-NP) have improved mucoadhesion compared to unloaded chitosan nanoparticles (CS-NP), suggesting that curcumin partly contributes to the mucoadhesion process. This may lead to an enhanced anticancer effect of curcumin when formulated in CUR-CS-NP. Our results show that CUR-CS-NP are taken up to a greater extent by colorectal cancer cells, compared to free curcumin. The prolonged contact offered by the mucoadhesion of CUR-CS-NP onto the cells resulted in a greater reduction in percentage cell viability as well as a lower IC50, indicating a potential improved treatment outcome. The formulation and free curcumin appeared to induce cell apoptosis in colorectal cancer cells, by arresting the cell cycle at G2/M phase. The superior anticancer effects exerted by CUR-CS-NP indicated that this could be a potential treatment for colorectal cancer

Induced pluripotent stem cells: history, properties and potential applications

The development of induced pluripotent stem cells (iPSCs) has been met with much enthusiasm and hailed as a breakthrough discovery by the scientific and research communities amidst the divisive and ongoing debates surrounding human embryonic stem cells (hESC) research. The discovery reveals the fact that embryonic pluripotency can be generated from adult somatic cells by the induction of appropriate transcriptional factor genes essential for maintaining the pluripotency. They provide an alternative source for pluripotent stem cells, thus representing a powerful new research tool besides their potential application in the field of regenerative medicine. In this review, the historical background of iPSCs generation will be discussed together with their properties and characteristics as well as their potential therapeutic applications

In silico prediction and validation of Gfap as miR-3099 target in the mouse brain

MicroRNAs are small non-coding RNAs that play crucial roles in the regulation of gene expression and protein synthesis during brain development. MiR-3099 is highly expressed throughout embryogenesis, especially in the developing central nervous system. Moreover, miR-3099 is also expressed at a higher level in differentiating neurons in vitro, suggesting that it is a potential regulator during neuronal cell development. This study aimed to predict the target genes of miR-3099 via in-silico analysis using four independent prediction algorithms (miRDB, miRanda, TargetScan, and DIANA-micro-T-CDS) with emphasis on target genes related to brain development and function. Based on the analysis, a total of 3,174 miR-3099 target genes were predicted. Those predicted by at least three algorithms (324 genes) were subjected to DAVID bioinformatics analysis to understand their overall functional themes and representation. The analysis revealed that nearly 70% of the target genes were expressed in the nervous system and a significant proportion were associated with transcriptional regulation and protein ubiquitination mechanisms. Comparison of in situ hybridization (ISH) expression patterns of miR-3099 in both published and in-house-generated ISH sections with the ISH sections of target genes from the Allen Brain Atlas identified 7 target genes (Dnmt3a, Gabpa, Gfap, Itga4, Lxn, Smad7, and Tbx18) having expression patterns complementary to miR-3099 in the developing and adult mouse brain samples. Of these, we validated Gfap as a direct downstream target of miR-3099 using the luciferase reporter gene system. In conclusion, we report the successful prediction and validation of Gfap as an miR-3099 target gene using a combination of bioinformatics resources with enrichment of annotations based on functional ontologies and a spatio-temporal expression dataset