A number of scientists and clinicians in Glasgow are currently participating in the National Lung Matrix Trial (NLMT), which aims to expand the use of stratified medicine approaches for the treatment of non-small cell lung carcinoma (NSCLC), by testing the efficiency of selected biomarker-targeted therapy combinations.
In the trial, NSCLC patients will be profiled using targeted genomic sequencing and assigned to one of 18 molecular cohorts depending on the presence or absence of specific mutations, and will be treated with drugs that target these particular mutant phenotypes. Successful drug-biomarker combinations will then be followed up in further investigations.
To better help the University's students understand the stratified medicine approaches that are being developed for cancer treatment, Horizon has volunteered the use of its X-MAN® Isogenic Cell Lines to recreate the trial in the lab. This multi-student project will test several of the drugs used in the NLMT on NSCLC, colon cancer and breast cancer cell lines that express biomarkers corresponding to different molecular cohorts, e.g. activated EGFR, activated KRAS.
Given the time intensive nature of gene engineering, the relatively straightforward and quick (<1 week) process of gRNA validation can save weeks of cell culture and hours of bench time. Further to this, a clear idea of gRNA activity will provide insights into how many clones need to be screened to identify a positive.
There are now a variety of methodologies available to scientists looking to compare and validate guide RNA activity. In this article we detail some of the most commonly used...
Much information about the role of specific genes in fundamental biological processes and the onset and progression of genetic disease has been gleaned by researchers having the ability to selectively alter the genomic composition of individual genes and study the consequences. This approach enables researchers to observe the effects of a specific mutation, SNP or deletion in combination with the added layers of regulation present within the cell, including post-translational modification, epigenetic changes associated with chromatin structure, and transcriptional mechanisms.
While the Nobel prize winning work of Capecchi, Evans and, and Smithies introduced the concept of manipulating the genome of mouse ES cells, the ability to manipulate a broader range of cell types, human cells in particular, remained a significant challenge for some time. The more recent discoveries of nuclease based targeting technologies like zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR) has greatly increased interest in genome editing and provided even more efficient platforms for achieving targeted genome modification. Despite the increases in efficiencies these technologies offer, there are still a wide range of factors that influence success and failure in genome editing.
While the scope of genome editing is very broad and includes whole organisms, this article will focus on the issues faced primarily by scientists attempting to modify the genome of immortalized cell lines. The ability to create isogenic cell lines in which the genome editing event is the sole differentiator between the phenotypes of two cells is a powerful tool. Despite all the recent developments and improvements in targeting platforms, certain challenges remain and the role played by the choice of cell line in order to achieve success cannot be understated.
It’s hard to keep up with the rapidly expanding world of CRISPR, and it’s starting to feel like CRISPR screens are being published every week, taking the technique from the cutting edge to the mainstream.
If you’d like to understand a bit more about CRISPR screens, here’s a number of fantastic publications that have really moved this technology forward...
Thanks to next generation sequencing (NGS), we are starting to understand the mutational changes that occur across the board in the cancer genome. With this knowledge comes potential – novel mutated genes and the proteins that they encode are candidates for prognostic markers and/or new drug targets. However, taking these initial findings all the way through to the clinic is an enduring challenge. RNAi technologies initially looked as if they might provide a new avenue for more effective drug discovery, but off-target effects and variable knockdown efficiencies make this a fraught approach unless stringently controlled.
The advent of gene editing using CRISPR–Cas9 — essentially re-purposing a primitive adaptive immune response in bacteria — provides a new and powerful tool to interrogate gene function on a genome-wide level. From our current vantage point, the contribution of this technology to drug discovery looks like it will be substantial, primarily because the off target effects appear to be fewer in CRISPR–Cas9 target ID screens.
In the article below, we go into a bit more detail about what CRISPR Screening is, and how we do it at Horizon Discovery.