Study of cellular delivery of siRNA and shRNA targeting bcr-abl in chronic myeloid leukemia using Tat derived peptide

Chronic Myeloid Leukemia is characterised by the formation of a fusion gene bcr-abl. The gene product BCR-ABL has deregulated tyrosine kinase activity that plays a direct role in the pathogenesis of the disease. Recently, use of siRNA in leukaemic cells has led to effective gene silencing of bcr-abl. Gene delivery systems like viral vectors, electroporation and lipid based vectors have showed varying efficiencies but are limited by their level of toxicity and immunogenicity. Developments in the field of Cell Penetrating Peptides have shown effective cellular uptake of nucleic acids and proteins by the CPPs in vitro and in vivo. Report from our lab has shown the use of CPP Tat along with membrane active peptide LK15 to improve the transfection efficiency of both Tat and LK15 peptides individually. Hence, this study will focus on the use of Tat-LK15 peptide to study the delivery of siRNA and shRNA plasmid in K562 cells and observe the BCR-ABL protein expression. Cellular uptake studies using Tat-LK15 based complexes of Cy5-labelled DNA and siRNA showed a concentration dependent uptake leading to increase in percentage transfected cells. Tat-LK15 based DNA complexes achieved 80% transfected cells (charge ratio of 2:1) while siRNA complexes resulted in a maximum of 60% (charge ratio of 3:1). However, Lipofectamine based DNA complexes did not show a concentration dependent increase in percentage transfected cells. Interestingly, Tat-LK15 based siRNA complexes showed a similar level of uptake and percentage transfected cells as that of Lipofectamine based siRNA complexes. Cellular uptake studies using confocal microscopy 4 hours post transfection, showed that when 1μg of DNA was transfected, the labelled DNA was primarily localised on the cell membrane. Interestingly, using 5μg of DNA led to increased intracellular localisation of the labelled DNA, but this observation was not made with Lipofectamine based complexes. The observation at 24 hours post transfection of Tat-LK15/labelled DNA complexes was of higher intensity when compared to that of Lipofectamine based DNA complexes. The reason for this is however not known. Interestingly, the cellular uptake profile using siRNA based complexes was different. At 4 hours post transfection, there was intracellular localisation of labelled siRNA. 24 hours post transfection, there was diffuse cytoplasmic localisation using lower concentration of siRNA whereas using higher concentration led to more high intensity punctate localisations within the cell. Similar observations were made for both Tat-LK15 and Lipofectamine based siRNA complexes.Gene silencing studies of Tat-LK15/shRNA plasmid complex resulted in 80% reduction in protein levels 96 hours post transfection for higher concentrations of shRNA plasmid treated. Similar level of reduction in BCR-ABL was observed with Lipofectamine based complex. Supporting evidence of reduction in mRNA levels was observed using qRT-PCR 48 hours post transfection. However, Tat-LK15/shRNA plasmid complexes led to around 80% of protein reduction 192 hours post transfection while Lipofectamine based complexes resulted in only 40% of protein reduction. Transfection using increasing concentrations of siRNA complexed to Tat-LK15 and Lipofectamine led to greater than 70% reduction in protein levels for most concentration ranges tested. This reduction in protein levels lasted only 48 hours post transfection. In conclusion, Tat-LK15 peptide could be used for shRNA plasmid and siRNA based delivery and could offer an efficient gene delivery model for studying RNAi.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Evaluation of aggregate and silicone-oil counts in pre-filled siliconized syringes: An orthogonal study characterising the entire subvisible size range.

Characterisation of particulates in therapeutic monoclonal antibody (mAb) formulations is routinely extended to the sub-visible size-range (0.1–10 μm). Additionally, with the increased use of pre-filled syringes (PFS), particle differentiation is required between proteinaceous and non-proteinaceous particles such as silicone-oil droplets. Here, three orthogonal techniques: Raster Image Correlation Spectroscopy (RICS), Resonance Mass Measurements (RMM) and Micro-Flow Imaging (MFI), were evaluated with respect to their sub-visible particle measurement and characterisation capabilities. Particle formation in mAb PFS solutions was evaluated with increasing polysorbate-20 (PS-20) concentrations. All three techniques provided complementary but distinct information on protein aggregate and silicone-oil droplet presence. PS-20 limited the generation of mAb aggregates during agitation, while increasing the number of silicone-oil droplets (PS-20 concentration dependant). MFI and RMM revealed PS-20 lead to the formation of larger micron-sized droplets, with RICS revealing an increase in smaller sub-micron droplets. Subtle differences in data sets complicate the apparent correlation between silicone-oil sloughing and mAb aggregates’ generation. RICS (though the use of a specific dye) demonstrates an improved selectivity for mAb aggregates, a broader measurement size-range and smaller sample volume requirement. Thus, RICS is proposed to add value to the currently available particle measurement techniques and enable informed decisions during mAb formulation development