Effects of pharmacological and genetic disruption of CXCR4 chemokine receptor function in B-cell acute lymphoblastic leukaemia.

B cell acute lymphoblastic leukaemia (B-ALL) cells express high levels of CXCR4 chemokine receptors for homing and retention within the marrow microenvironment. Bone marrow stromal cells (BMSC) secrete CXCL12, the ligand for CXCR4, and protect B-ALL cells from cytotoxic drugs. Therefore, the therapeutic use of CXCR4 antagonists has been proposed to disrupt cross talk between B-ALL cells and the protective stroma. Because CXCR4 antagonists can have activating agonistic function, we compared the genetic and pharmacological deletion of CXCR4 in B-ALL cells, using CRISPR-Cas9 gene editing and CXCR4 antagonists that are in clinical use (plerixafor, BKT140). Both genetic and pharmacological CXCR4 inhibition significantly reduced B-ALL cell migration to CXCL12 gradients and beneath BMSC, and restored drug sensitivity to dexamethasone, vincristine and cyclophosphamide. NOD/SCID/IL-2rγnull mice injected with CXCR4 gene-deleted B-ALL cells had significant delay in disease progression and superior survival when compared to control mice injected with CXCR4 wild-type B-ALL cells. These findings indicate that anti-leukaemia activity of CXCR4 antagonists is primarily due to CXCR4 inhibition, rather than agonistic activity, and corroborate that CXCR4 is an important target to overcome stroma-mediated drug resistance in B-ALL.

CRISPR-Cas9 in cancer therapeutics.

Cancer is a disease mainly caused by an accumulation of mutations in cells. Consequently, correcting those genetic aberrations could be a potential treatment strategy. The traditional route for cancer drug development is tedious, laborious, and time-consuming. Due to target identification, drug formulation, pre-clinical testing, clinical testing, and regulatory hurdles, on average, it takes 10-15 years for a cancer drug to go from target discovery to a marketable oncology drug. The advent of CRISPR-Cas9 technology has greatly expedited this procedure. CRISPR-Cas9 has single-handedly accelerated target identification and pre-clinical testing. Furthermore, CRISPR-Cas9 has also been used in ex vivo editing of T-cells to specifically target tumor cells. In this chapter, we will discuss the various ways in which CRISPR-Cas9 has been used for the betterment of the cancer drug development process. Additionally, we will discuss various ways in which it is currently being used as therapy and the drawbacks which restrict the use of this groundbreaking technology as direct therapy.

The evolution and history of gene editing technologies.

Scientific enquiry must be the driving force of research. This sentiment is manifested as the profound impact gene editing technologies are having in our current world. There exist three main gene editing technologies today: Zinc Finger Nucleases, TALENs and the CRISPR-Cas system. When these systems were being uncovered, none of the scientists set out to design tools to engineer genomes. They were simply trying to understand the mechanisms existing in nature. If it was not for this simple sense of wonder, we probably would not have these breakthrough technologies. In this chapter, we will discuss the history, applications and ethical issues surrounding these technologies, focusing on the now predominant CRISPR-Cas technology. Gene editing technologies, as we know them now, are poised to have an overwhelming impact on our world. However, it is impossible to predict the route they will take in the future or to comprehend the full impact of its repercussions.

Preparation and characterization of extracellular vesicles.

Extracellular vesicles (EVs) are heterogeneous membranous vesicles secreted by every cell type and offer significant potential in therapy and diagnostics. Differential ultracentrifugation is the gold standard for EV isolation, although other techniques including, polyethylene glycol (PEG) precipitation, immunoprecipitation, size exclusion chromatography, and immuno-isolation approaches are common. Purified EVs can be characterized based on their physical characteristics, biochemical composition, or cell of origin. For size and concentration measurement, nanoparticle tracking analysis (NTA), dynamic light scattering (DLS), and electron microscopy are commonly employed methods. Biochemical analyses of EVs are typically performed using flow cytometry, immunoblotting, or proteomic investigation. Based on tissue of origin, EVs have specific markers that can be used to isolate and purify specific cell-associated EVs using an affinity selection approach. Despite existence of several methods for isolation and characterization, major limitations associated with each method hinder the progress of the field. Evolving concepts in EV biology possess great promise for better isolation and characterization leading to a better insight of biological function and have immense clinical implications. In this review, we discuss recent advancements in EV isolation and characterization approaches.