In a new review article, just published in the New England Journal of Medicine, Matthew Porteus, MD, PhD, head of the Porteus Lab in the division of stem cell transplantation and regenerative medicine at Stanford University School of Medicine in California, broke down the types of genome editing that are being explored across various clinical trials, the ways in which chimeric antigen receptor (CAR) T-cell therapy (CAR-T) will likely be optimized, and the differences between nonhomologous end-joining (NHEJ) and homology-directed repair (HDR)-based DNA repair mechanisms.1

Dr Porteus, MD, PhD, directs research on genome engineering and has written papers on the topic of gene editing, with a focus on the various platforms for editing that are already available or are in development. These nuclease-based platforms have included transcription activator-like effector nucleases (TALEN), zinc finger nucleases, and CRISPR–Cas9.

In his most recent review, Dr Porteus wrote that the editing platform shouldn’t really be called CRISPR–Cas9. Instead, he reasoned, because it uses only 2 components of the bacterial immune system for genome editing, it should be coined the Cas9–gRNA system. The methods of getting the desired genetic material into a cell can vary; most of the trials in cancer that used or will use genome editing are relying on mRNA as a delivery mechanism (as opposed to, say, viral vectors). And, although Dr Porteus suggested that mRNA may be a better nuclease delivery method than plasmid DNA, he pointed out that “mRNA can induce an antiviral type I interferon response.” What’s more, he added, “prolonged expression of a nuclease or expression of a nuclease with low specificity can result in sustained activation of the p53 pathway, thereby triggering cell-cycle arrest and apoptosis.”

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Dr Porteus is also a founder of CRISPR Therapeutics and published a prior paper with colleagues that revealed that a majority of people have preexisting immunity to the Cas9 enzyme.2 There appear to be more subjects included in the Nature Medicine paper than were analyzed in an earlier bioRxiv version, and there were differences in the assays used for T-cell detection across these 2 versions of the paper,3 among other differences, but the final conclusion about human reactivity to the nucleases remained relatively unchanged.

Of the 10 sponsors of the genome-editing trials in cancer that Dr Porteus highlighted in the most recent review, 5 sponsors are running trials in China, and 5 sponsors are conducting studies in either Europe or the United States.1 The malignancies being studied across these studies include acute lymphoblastic leukemia and lymphoma, T-cell acute lymphoblastic leukemia, blastic plasmacytoid dendritic cell neoplasm, acute myeloid leukemia, solid tumors, multiple myeloma, bladder cancer, prostate cancer, and renal cell carcinoma.

The trials in cancer that involve ex vivo gene editing that will begin in the United States and Europe in the coming years harness NEHJ-based gene knockout to make CAR-T more potent. Other improvements to CAR-T cell therapy based on gene editing are under investigation, and these include development of universal CAR-T cells (gene editing to reduce their immunogenic components), as well as some gene modifications to prevent T-cell exhaustion.

And, of course, better nucleases likely mean better editing control. But this is not limited to improvements to the Cas9 orthologs themselves; Dr Porteus wrote that highly specific Cas9 variants got even more specific when “delivered as a ribonucleoprotein complex.”

Despite the potential of these advances in editing, and the “hundreds of genetic diseases” that could feasibly be “cured” through gene correction, Dr Porteus also issued caveats about DNA editing. For one, he wrote, “there are no data to provide guidance as to what is a safe level of off-target indels for either ex vivo or in vivo uses of genome editing.” The assays that are currently used to detect off-target mutations are not sensitive enough, nor can they properly measure the “functional consequences” of off-target changes. And, he seemed to reference another recent paper covering CAR-T cell therapy manufacturing when he mentioned that if researchers use a single clone to grow vats of cells, there is a chance that during expansion, the product could be “dominated by a cell with a spontaneous mutation in a tumor-suppressor gene or by an oncogene that is selected for expression.”


  1. Porteus MH. A new class of medicines through DNA editing [published online March 6, 2019]. New Engl J Med. doi: 10.1056/NEJMra1800729
  2. Charlesworth CT, Deshpande PS, Dever DP, et al. Identification of preexisting adaptive immunity to Cas9 proteins in humans. Nat Med. 2019;25:249-254.
  3. Charlesworth CT, Deshpande, PS, Dever DP, et al. Identification of pre-existing adaptive immunity to Cas9 proteins in humans. bioRxiv. Published January 5, 2018. doi: 10.1101/243345