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January 15, 2011 (Vol. 31, No. 2)

Finessing Gene Editing in Human Cells

Genetically Defined X-Man Disease Models Find Niche in Personalized Medicine Applications

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    Figure 1. Genetically defined X-Man cell lines and their applications

    The output of high-throughput genome sequencing technologies has been accelerating at an exponential rate and, consequently, the full-scope of human genetic variation is now within our reach. The technical bottleneck is now moving to “what does it all mean?” This crucial question is the challenge of functional genomics, an increasingly important field that will need both advances in human genetic manipulation techniques and large consortium-based research efforts to decipher how specific genetic variations predispose or accelerate human pathogenic disorders.

    In pursuit of these aims, Horizon Discovery  is commercializing (to biotech, pharma, and diagnostic companies) or providing in open access (to academic research organizations) a gene-editing platform called Genesis.

    This technique is built on the Nobel Prize winning discovery that homologous recombination (HR) can be harnessed to perform precise genome alterations in mice. Genesis takes this technique many steps further to permit efficient gene editing in any pre-established and differentiated human cell line, which in contrast to mouse ES cells, have low rates of homologous recombination. This is achieved using recombinant adeno-associated (rAAV) vectors which, due to their ssDNA genome, activate HR without resorting to causing dsDNA breaks in the genome, typical of zinc-finger nucleases (ZFNs) and meganucleases.

    Moreover, because dsDNA breaks are preferentially repaired by error-prone non-homologous end-joining pathways, ZFNs are more appropriate for performing loss-of-function gene knockouts. In contrast, rAAV can readily perform both knockout and gain-of-function knockins, enabling the modeling of subtle SNPs or activating point-mutations that are more commonly the targets of drug discovery programs.

    To date, Horizon has used Genesis primarily to generate a large and expanding panel of genetically defined X-Man (mutant and normal) disease models. These are matched pairs of cell lines, wherein one harbors a cancer-associated mutation in an endogenous gene, just as it occurs in real patients, and the other is an otherwise genetically identical cell line except that is carries a normal version of that gene.

    These isogenic disease models provide a definitive means to understand disease biology and develop new targeted therapies to specific patient populations.

    One of the most exciting applications of X-Man cell lines is to predict, prior to entering clinical trials, which patients will benefit most from a new targeted therapy. The benefits of tailoring the right drug to the right patients in clinical trials will inevitably lead to faster approvals, more ethical patient accrual, and less chance of drug effects being missed in larger unselected patient cohorts. Such information is essential in order for healthcare agencies to ensure that only patients with a rational and diagnosable predictive biomarker are a given a specific medication.

    Colon cancer patients with K-Ras mutations were recently assessed in retrospective and prospective studies to be nonresponsive to EGFR-therapies Erbitux and Vectibix. As a result, testing of K-Ras mutational status is now mandatory prior to prescription of these agents. It should be noted, however, that there are several different mutational variants of K-Ras and currently a patient carrying any variant K-Ras is excluded from anti-EGFR therapy in both the EU and the U.S.

    In an attempt to provide greater resolution to the influence of specific K-Ras mutational biomarkers, and in response to a small number of clinical anomalies to this resistance profile, Horizon’s co-founder, Alberto Bardelli, set out to test two X-Man cell lines harboring the most predominant K-Ras mutations (G13D or G12V) for their response to Erbitux.

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