1. Kmiec, Eric PhD

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It is now possible to manipulate the human genome like never before. And believe it or not, the tool that has revolutionized biomedical research has been operational in bacterial cells since the beginning of time; in other words, it's been sitting right in front of us, ready for action.

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Clustered, regularly interspaced short palindromic repeats have thankfully been assigned the acronym, CRISPR. When combined with a partner enzyme, Cas9, another tool provided by bacteria, CRISPR can seek out DNA sequences with extraordinary precision, bind tightly and initiate a specific double strand cut, akin to the action of a pair of molecular scissors.


While each of these steps could be executed previously, the simplicity of design, the reduction in experimental time, and the extraordinary efficiency with which the scissors act, the result is simply unprecedented. CRISPR/Cas9 is a rare instrument, one of the few transformative molecular tools ever developed, and among the most important.


CRISPR systems entered the mainstream of biomedical research in the summer of 2012, when the Doudna and Charpentier research teams reported that they had adapted CRISPR for use in human genome engineering. The design of CRISPR to target any known DNA sequence is remarkably simple, and today, most research laboratories interested can design their own CRISPR using publically available bioinformatics databases.


By far, the most common application of CRISPR is the creation of a gene knockout in which the coding region of a human gene is disabled and rendered nonfunctional. Another practical use is combining the cleavage activity of a specific CRISPR system with genetic information provided by a donor DNA molecule, most often in the form of single-stranded oligonucleotides, to correct single base mutations to patch or replace malfunctioning sections of a gene that account for a variety of inherited diseases.


Other uses include modulation gene expression through the use of an inactivated Cas9 protein and the insertion of fluorescent tags within the human genome so that genetic migration can be monitored. In a different arena, CRISPR/donor DNA complexes are being developed for treating genetic diseases such as sickle cell anemia, Gaucher's disease, Criglar-Najjar syndrome or even cystic fibrosis. In addition, the well-publicized gene editing of the mosquito genome is aimed at eliminating the spread of malaria not only in endemic populations but worldwide.


Molecular Basis of Oncogenesis

But, what about using CRISPR for cancer research or even cancer therapeutics? A colleague of mine told me about a year ago that she has seen technologies come and go, and the chances of seeing CRISPR used successfully to treat human cancer or impact cancer research significantly is about the same as a monkey playing Mozart. Well, maybe.


Acting in concert, two CRISPR complexes can catalyze chromosomal translocations that are foundational in the development of various types of leukemia and have been recapitulated successfully in cell lines. CRISPR is now being used to reconstruct chromosomal translocations with high efficiency and accuracy enabling the generation of cell models harboring the same alterations that define tumor cells.


Appropriate CRISPR systems have successfully reconstructed the t (11:22)/EWS-FL1 translocation involved in Ewing sarcoma; the t (8:11)/RUNX1-ETO translocation responsible for one form of acute myeloid leukemia; and the t(5:6)/CD74-Ros1 translocation involved in lung adenocarcinoma. In these and other cases, the genomic rearrangement creates a fusion gene that enables the production of an oncogenic protein; thereby providing an important testing system for pharmaceutical companies engaged in anticancer drug screening. CRISPR systems are also helping to elucidate the pathway and fate of single cells after they gain the capacity to migrate and invade at distal sites by creating a series of molecular barcodes in single cell lineages.


Clinical Applications in Cancer

By and large, the closest to the clinical application of gene editing centers on its use in T cells, because these cells are accessible, can be isolated from a patient and genetically altered ex vivo and reintroduced into the patient with or without expansion. This effort is exploring the use of CRISPR for cancer immunotherapies, specifically chimeric antigen receptor T cells (CAR T cells). CARs are proteins that provide the recognition activity of a specific antigen on tumor cells and gene editing approaches using CRISPR, among other techniques, and are being used to enhance the recognition and cell killing capacity of T cells for those antigens. The objective is to improve T cell persistence by genetically altering the human genome so it enhances immune-recognition and elevates activity by targeting tumor cells.


Recently and importantly, scientists at the University of Pennsylvania have begun an experiment in human patients to target refractory cases of myeloma, sarcoma, and melanoma. The experimental strategy is, in principal, similar to the CAR T approach where gene(s) are modified (disabled) to more effectively target the tumor cell. Again, the modification takes place ex vivo and the genetically altered T cells are then infused back into the patient.


The first step in this clinical approach is a safety trial that will begin shortly. The significance of this trial cannot be understated because it received approval from the Recombinant Advisory Committee, the federal ethics panel of the NIH. This panel was established over 40 years ago to undertake fundamental reviews of state-of-the-art and even controversial experimentation on the human genome.


CRISPR is also being considered as an experimental strategy for the treatment of lung cancer on two parallel tracts. The first is molecular surgery that aims to correct or destroy a mutated epidermal growth factor receptor (EGFR) gene (EMBO Mol Med 2016: 8; 83-85). The second approach aims to use CRISPR to disable genes that confer varying degrees of resistance to chemotherapy, developed over the course of tumorigenesis and extended treatment.


These studies hold promise for using gene editing in combinatorial therapy coupled with well-established chemo or radiation treatment regimens. The efficiency with which the CRISPR/Cas9 operates to disable either alone or as multiple systems renders its use as a particularly attractive supplemental approach for the development of novel treatments.


Barriers to Success

As with any innovative biomedical strategy, there is always a "rest of the story." For gene editing applications, there are several obstacles that need to be overcome prior to successful clinical implementation.


First is the need to develop an effective delivery method(s) so that enough of the gene editing tool(s) is delivered in vivo to the targeted tissue or organ. There are new developments of synthetic carriers, such as ribonucleoprotein-nanoparticle complexes, which might be an attractive resource, while hydrodynamic delivery of gene editing molecules is currently being tried for both liquid and solid tumors.


Second, there is always a possibility that a specific CRISPR/Cas9 acts nonspecifically, in that off-target sequences could be cleaved and, through the natural heterogeneous resealing activity, inappropriate modifications could be made to distal DNA sequences. Many workers in this field have been wrestling with this problem and variations of the Cas9 protein, created by reducing or removing its nuclease activity, appear to be improving the already high-level specificity. No doubt the field is also limited now by the lack of predictable yet robust assays to detect such off-site mutagenesis, so much so that a confident answer regarding off-site mutagenesis might always remain elusive.


Finally, it will be imperative that clinical approaches using gene editing include an evaluation of all cells exposed to these molecular tools. In our own laboratory, we worry about the cells that are not converted and we are trying to evaluate what happens to them; have they been genetically altered but not converted and at some point will they develop an oncolytic lineage as a regrettable form of collateral damage? These are questions that can be answered. Without a doubt, this is an exceedingly exciting time for the field of gene editing in cancer research, full of innovation and spectacular progress.

Eric Kmiec, PhD. ERI... - Click to enlarge in new windowEric Kmiec, PhD. ERIC KMIEC, PHD, is the Director of the Gene Editing Institute at the Helen F. Graham Cancer Center & Research Institute at Christiana Care Health System in Newark, Del.