Patricia F. Fitzpatrick Dimond Ph.D. Technical Editor of Clinical OMICs President of BioInsight Communications

The Promise of a Big Payoff Seems Very Likely

The gene-editing technology known as CRISPR, short for clustered regularly interspaced short palindromic repeats, has raced out of the research laboratory to reach the clinic in record time, as NIH’s Recombinant DNA Advisory Committee green-lighted the first U.S. clinical trial of the technology in the U.S. in June. Trial investigators propose to use CRISPR/Cas9 to help augment cancer therapies that rely on enlisting a patient’s T cells.    

The CRISPR system, first identified in bacteria and later shown to serve as a primitive form of immunity in these organisms, may have the potential to resurrect gene therapy as a valid approach for treating human diseases.

Significant failures resulted after earlier efforts to exploit gene editing as a therapeutic tool by other methods, notably the death of 18-year old Jesse Gelsinger in 1999 in an attempt to correct ornithine transcarbamylase deficiency, a rare metabolic disorder.

The intention was to deliver corrective genes using an adenoviral vector, but it apparently produced a massive and deadly immune response, including jaundice, a blood-clotting disorder, kidney failure, lung failure, and brain death. Medical scientists at the University of Pennsylvania where the death occurred are about to try again using CRISPR technology to treat patients with several cancers that have not responded to other forms of treatment.

The CRISPR Patent Wars Continue

Work by innovative researchers has produced remarkable progress in adapting CRISPR technology to mammalian cells. But spectacular patent wars continue as biotech companies compete to get the first CRISPR system to the therapeutic market, and academic institutions where scientists developed various incarnations of the technology continue to do battle.

Most recently Editas Medicine entered into an exclusive license agreement with Massachusetts General Hospital to access intellectual property and technology related to high-fidelity Cas9 nucleases and Cas9 protospacer adjacent motif variants.  

As of February, the U.S. Patent and Trademark Office had issued 28 patents with claims to CRISPR and/or Cas9, including a “robust” portfolio of 13 CRISPR patents to the Broad Institute, MIT, and affiliated groups for inventions from Dr. Zhang and his lab.

Combatants in the patent wars include publicly held Intellia Therapeutics and CRISPR Therapeutics.  Intellia, which owns its CRISPR rights through Jennifer Doudna, Ph.D., raised $108 million in its May IPO.  

UC Berkeley’s Dr. Doudna and her colleagues, in their 2012 paper “A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Vacterial Immunity” 1 broke CRISPR ground by describing its therapeutic potential in Science.

CRISPR Mechanisms Elucidated

All of this patent mayhem began when a well-prepared mind belonging to Francisco Mojica, Ph.D., a scientist at the University of Alicante in Spain, made a fundamental observation. He and his colleagues recognized that what previously had been reported as disparate repeat sequences in bacterial genomes shared a common set of features, now known to be characteristic of CRISPR sequences.2

In a later report in the Journal of Molecular Evolution,3 these scientists proposed that CRISPRs, consisting of repeating sequences of genetic code interrupted by spacer sequences, comprise the detritus of genetic codes from past invaders such as bacteriophage. These sequences serve part of the bacterial immune system, a genetic memory allowing the cell to detect and destroy invaders. 

And in 2007, a team of scientists led by Philippe Horvath, Ph.D.,4 provided experimental proof that Dr. Mojica’s theory was correct.

Another key finding in elucidating the mechanism of natural CRISPR/Cas9-guided interference came from the research of Elitza Deltcheva, Ph.D., and colleagues working in the laboratory of Emmanuelle Charpentier, Ph.D., at the Laboratory for Molecular Infection Medicine Sweden (MIMS).5 The researchers, in sequencing small RNA in Streptococcus pyogenes, which has a Cas9-containing CRISPR/Cas system, discovered that in addition to CRISPR RNA (crRNA) the bacterium contained a second small RNA they called trans-activating CRISPR RNA (tracrRNA). They showed that tracrRNA forms a duplex with crRNA, and it is this duplex that guides Cas9 to its targets.

In January 2013, Le Cong and colleagues working at the Broad Institute of MIT and Harvard in Dr. Zhang’s lab reported the first successful demonstration of Cas9-based genome editing in human cells.6

Prashant Mali et al., working in George Church’s lab at Harvard University, reported similar findings in the same issue of Science,7 showing that Cas9 could be targeted to a specific location in the human genome and then excise the DNA at that locus, which could then be repaired by inserting a new stretch of DNA of the researchers choosing.

Competitive technologies, according to Science, which enshrined CRISPR as its technology of the year in 2015, include zinc finger nucleases and transcription activator-like effector nucleases (TALENs) that can also precisely change chosen DNA sequences. But CRISPR has proven so easy and inexpensive that Dana Carroll of the University of Utah, a developer of zinc finger nucleases, commented that CRISPR technology has enabled the “democratization” of gene targeting

The picture that has emerged from the multiple laboratories regarding CRISPR describes a system that protects bacteria and archaea from both phage infection and plasmid invasion, and research has confirmed that it can perform gene edits in a variety of cell types. These loci harbor short sequences of phage and plasmid origin known as “spacers” that specify the targets of CRISPR/Cas immunity. Requisite components of CRISPR systems include two DNA-editing tools—the enzyme Cas9, which cuts DNA at specific loci, and guide RNA (gRNA), a small piece of predesigned RNA sequence about 20 bases long located within a longer RNA scaffold. The scaffold part binds to DNA and the predesigned sequence “guides” Cas9 to the right part of the genome, ensuring that the enzyme cuts at the right point in the genome.

CRISPR Cures for Human Diseases

Whether CRISPR technology will cure human diseases remains to be seen. On June 21, an advisory committee at the NIH approved a proposal to use CRISPR/Cas9 to help augment cancer therapies that rely on enlisting a patient’s T cells.

During this study, intended to assess CRISPR technology safety, investigators will remove T cells from 18 patients with melanoma, multiple myeloma, and sarcoma whose cancers have stopped responding to therapies.

The two-year study, funded by a $250-million immunotherapy foundation established by former Facebook president Sean Parker, will be conducted at three sites that are members of the Parker Institute—The University of Pennsylvania, the University of California, San Francisco, and the University of Texas MD Anderson Cancer Center in Houston.

These investigators propose to make three modifications in patients’ T cells prior to reinfusing them, one of which includes engineering the cells to express an affinity-enhanced T-cell receptor (TCR) that recognizes a naturally processed peptide shared by the cancer antigens NY-ESO-1 and LAGE-1. The customized TCR will be inserted into cells using a virus. CRISPR will be used to disable the existing TCR to focus the altered cells on targeting tumors instead of other nontumor targets.

Because two genes code for this receptor, this involves disabling two genes. CRISPR will also be used to neutralize the PD-1 gene, which expresses a protein on T-cell surfaces that many cancers can turn off, thereby blocking T-cell antitumor attacks.

And Chinese scientists are about to start a clinical trial using CRISPR-modified cells in lung cancer patients this month. Led by Lu You, M.D., an oncologist at Sichuan University’s West China Hospital in Chengdu, the study will enroll patients with metastatic non-small-cell lung cancer (NSCLC) in whom chemotherapy, radiation therapy, and other treatments have failed.

Dr. You’s team will extract T cells from enrolled patients, then use CRISPR/Cas9 technology to knock out the PD-1 gene, the protein product which blocks the cells’ capacity to attack cancer cells and that normally functions to prevent T cells from attacking healthy cells.   

Once expanded in the laboratory, the edited cells will be returned to the patient. The engineered cells will circulate and, the team hopes, home in on the cancer, says Dr. You. The planned U.S. trial similarly intends to knock out the gene for PD-1, and it will also knock out a second gene and insert a third before the cells are reintroduced into the patient.

Importantly, in assessing the technology’s safety for clinical use, the U.S. scientists, whose ambitious approach to gene editing involves three changes, will look for off-target effects, or signs that cuts in the wrong places have occurred, potentially creating genomic changes that could create or trigger cancer.

CRISPR Developments Continue

All of this remains risky business. The discovery of CRISPR systems and development of enhancements also has spawned a new generation of genome engineering because it enables previously undoable feats. Alexis Komor et al., writing in the May 19, 2016 issue of Nature8 and working in David R. Liu’s lab in the department of chemistry and chemical biology at Harvard University, reported the development of “base editing,” which enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring a double-stranded DNA (dsDNA) backbone cleavage or a donor template.

These authors engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a gRNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides, and can “efficiently correct” a variety of point mutations relevant to human disease, the authors said.

While backed by some truly glitzy science, the highly focused efforts described here may generate safe gene therapy treatments for intractable human diseases—in addition to immense legal fees.

1. Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. (2012). A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821.
2. Mojica FJ, Díez-Villaseñor C, Soria E, Juez G (2000). Biological significance of a family of regularly spaced repeats in the genomes of Archaea, Bacteria and mitochondria. Molecular Microbiology 36, 244–246.
3. Mojica FJ1, Díez-Villaseñor C, García-Martínez J, Soria E. (2005). Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements. Journal of Molecular Evolution 60, 174–182.
4. Barrangou R, Fremaux C, Deveau H, Richards M, Boyaval P, Moineau S, Romero D A, Horvath P. (2007). CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712.
5. Deltcheva E, Chylinski K, Sharma CM, Gonzales K, Chao Y, Pirzada ZA, Eckert MR, Vogel J, Charpentier E. (2011). CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607.
6. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. (2013). Multiplex genome engineering using CRISPR/Cas systems.  Science 339, 819–823.
7. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM. (2013). RNA-Guided Human Genome Engineering via Cas9. Science 339:823–826. 
8. Komor AC, Kim YB, Packer MS, Zuris JA, Liu DR. (2016). Programmable editing of a target base in genomic DNA without double-stranded DNA cleavage. Nature 533:420–424.


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