Authored by Debbie King, Contributor, Cell Culture Dish

Previously, I wrote an article summarizing the CRISPR/Cas9 system (CRISPR short for Clustered Regularly Interspaced Short Palindromic Repeats) and its applications. This prokaryotic defense mechanism (first discovered in 1987) which cleaves the DNA of invading phages and plasmids has revolutionized how we perform genetic modifications in eukaryotic cells since it was first published in 2013. It has enabled researchers to use the Cas9 ‘molecular scissors’ to cut DNA at a specific locus as directed by a guide RNA sequence. The technology has been rapidly accepted by researchers worldwide. For example, at the International Society for Stem Cell Research (ISSCR) conference this year in San Francisco CA, 90 of the 504 posters presented mentioned the CRISPR/Cas9 technology. That’s 18% of all the poster presentations spanning many different cell types and disciplines. It is amazing to see such a quick adoption of a technique in just three short years but certainly not surprising as researchers continue to be excited about the potential of CRISPR-based gene editing. It is a game-changer in the molecular toolbox, enabling efficient, cost-effective, precision gene editing that was simply not attainable before.

The potential of gene editing combined with induced pluripotent stem cells has also changed the way we approach human disease treatment so it was not unexpected to see ISSCR talks focused on translating stem cell therapies to the clinic. One of the talks on the first day of the conference was presented by the ISSCR Ethics Committee on the ‘Ethical Implications of Genome Editing Technologies’. A diverse group of individuals ranging from scientists, lawyers as well as patient advocates acknowledged the challenge in regulating these technologies across different countries with vastly different governing agencies. The takeaway for me was that openness in dialog between scientists and government is critical to move cell therapies forward safely and the debate over stem cell clinical regulations will continue to be a major focus for the field in the coming years as therapies using gene-editing technologies move to clinical trials.

First ‘In-Human’ CRISPR Clinical Trial

In the race towards the clinic with CRISPR/Cas9-modified human cells, it appears that China is leading the way. A team led by Lu You, an oncologist at Sichuan University’s West China Hospital in Chengdu, received ethical approval to test the cells in people with lung cancer on July 6, 2016 (Cyranoski, 2016) with the clinical trial initiated in August 2016. While the proposal submitted by a team at the University of Pennsylvania (UPenn) led by Carl June was approved by the Recombinant DNA Advisory Committee (RAC) at the US National Institutes of Health (NIH) ahead of the Chinese team on June 21, 2016, it has yet to receive the green light from medical centers where the trial would be conducted, the Food and Drug Administration (FDA), and the various university review boards of the researchers involved in the study (Kaiser, 2016).

In Phase I of a clinical trial, the aim is to test a new drug or treatment in a small group of individuals to evaluate its safety, determine a safe dosage range and identify any side effects. The Chinese trial will include ten patients diagnosed with metastatic non-small cell lung cancer that is non-responsive to conventional therapies (chemotherapy, radiation, etc). Immune cells called T cells play a key role in cell-mediated immunity and enhancing their function is critical to immunotherapy (Su, 2016). In the trial, T cells will be extracted from the patient’s blood and CRISPR/Cas9 will be used to disrupt the gene PD-1 which encodes the programmed death-1 (PD-1) receptor. PD-1 expression on the cell surface of activated T cells is part of what is known as ‘immune checkpoint regulation’. Immune checkpoints are molecules in the immune system that either turn up (stimulatory molecules) or turn down the immune response (inhibitory molecules) (Pardoll, 2012). PD-L1 is an inhibitory checkpoint ligand expressed on the surface of most cells and functions through binding of PD-1 receptor on activated T cells, preventing them from inappropriately attacking healthy cells after an immune response has been launched. However, cancer cells have hijacked this system by acquiring the ability to also express PD-L1, essentially hiding from T cell attacks. Scientists hope that reduced gene expression of PD-1 by the patient’s T cells will allow them to seek out these cancer cells, once reinfused back into the patient, because they will not be as susceptible to false inhibitory cues. Critics of the trial are concerned that these engineered T-cells could cause deleterious autoimmune responses since the endogenous immune checkpoints have been removed. However, there are FDA-approved drugs like Merck & Co.’s drug Keytruda™ (therapeutic antibody) – approved for lung cancer treatment in 2015 (USDA, 2015) – that also target the PD-1/PD-L1 pathway but have not resulted in significant autoimmune responses in patients. Also of concern are off-target gene edits, at other locations in the genome, known to occur when using CRISPR/Cas9. Because of this, biotechnology company Chengdu MedGencell will validate the T cells to confirm the correct gene edit of the PD-1 and to ensure there are no unintended genomic changes (Cyranoski, 2016). The researchers will test three cell dosage regimens with the ten patients and will also monitor markers in the blood of the patients, which would indicate whether the treatment is working.

The UPenn team has plans for a two year trial intending to treat 18 patients with myeloma, sarcoma, or melanoma who are no longer responding to existing treatments at three facilities: UPenn, the University of California (San Francisco) and the University of Texas MD Anderson Cancer Center (Houston). In addition to disrupting the PD-1 gene using CRISPR/Cas9 (as above), they will engineer the patient’s T cells using a lentiviral vector to express the receptor for NY-ESO-1. NY-ESO-1 is an antigen expressed by tumor cells that is not present on most healthy cells (Kaiser, 2016). The addition of the NY-ESO-1 receptor would direct the T cells to attack a patient’s NY-ESO-1-displaying tumor cells. The researchers also plan to knock out two gene segments of the endogenous T cell receptor (TCR), TCRA and TCRB genes – which control T-cell receptor alpha and beta chains, respectively – so that the engineered NS-ESO-1 receptor will be more effective.

Other clinical trials may not be far behind: Editas Medicine (NASDAQ: EDIT) in Cambridge, Massachusetts, is slated to use CRISPR/Cas9 in a clinical trial as soon as 2017 for treatment of Leber Congenital Amaurosis (LCA10), a rare form of blindness that affects the retinal cells. Many, myself included, thought Editas Medicine would conduct the first CRISPR clinical trial but RAC has not yet reviewed a proposal from the company.

CRISPR-edited Crops Not Considered GMO

There have also been some important developments on the agricultural front with regards to CRISPR/Cas9. On September 22, 2016, Monsanto, one of the agriculture giants, solidified licensing of the technology for use in seed development from the Broad Institute (Begley, 2016). This is the first license the institute has issued to a company for use in agriculture. The benefits of gene editing for crops include accelerated crop growth, resistance to pests and inclement weather, and increased nutritional benefit. However, with this license from the institute come some important restrictions for Monsanto:

• CRISPR/Cas9 cannot be used to initiate gene drives, a controversial technique where a genetic change is forced in an organism that is inherited and propagated in subsequent generations. Experimentation with this technology in the laboratory (closed system) has shown many unpredictable results. The impact of releasing gene drives in the wild raises major bioethical concerns because we do not understand how to control them.
• The company is barred from creating sterile seeds.
• CRISPR/Cas9 cannot be used for research and development on the tobacco plant related to smoking (i.e. making them more tolerant to environmental changes or pests, which could increase the yields).

Monsanto believes the technology will be much more powerful than the traditional technique behind GMOs (genetically modified organisms), which was imprecise and often took years to change a crop’s trait (Begley, 2016). CRISPR technology for altering crop genomes is even more attractive now because the US Department of Agriculture (USDA) has opted not to regulate CRISPR/Cas9-modified plants in the same way as conventional GMOs (Brodwin, 2016). The distinction lies in the fact that CRISPR-edited crops do not contain any ‘introduced genetic material’ or foreign DNA, which is the case with most GMOs. Altering the DNA sequence by changing a single nucleotide in a gene using CRISPR/Cas9 is a far cry from introducing a bacterial gene into a plant’s genome.

One such CRISPR/Cas9-engineered crop likely to be available in the next five years is DuPont Pioneer’s waxy corn (Zea mays) hybrid. Corn hybrids lacking the gene Wx1 are able to produce large quantities of a starch called amylopectin. This specialty grain is milled into starch, which is used in a variety of processed foods, adhesives and high-gloss paper. Traditionally, breeders had to seek out corn hybrids that have a naturally occurring mutation that makes Wx1 non-functional, however, these plants tended to have reduced yield. With CRISPR/Cas9, Wx1 can be directly targeted to render it non-functional while maintaining crop yields, a feat that could likely be accomplished through natural breeding but would take many generations to achieve. The USDA’s Animal and Plant Health Inspection Service (APHIS) acknowledged that CRISPR/Cas9 has been used to accelerate traditional breeding practices and therefore does not consider next-generation waxy corn as regulated by USDA Biotechnology Regulatory Services (in response to Pioneer’s “Regulated Article Letter of Inquiry”).

CRISPR Closing Remarks

In a relatively short time frame for a new technology, CRISPR/Cas9 has revolutionized the way we do science. Never before have genetic modifications been achieved with such precision and ease. Because the power of this technique, advances in the field of Gene Therapy have been swift, leading to the first ever in-human clinical trial three short years after the technique was used first in eukaryotic cells. It has also proving useful in the plant science community as a powerful tool for the improvement of agricultural crops particularly with the new USDA rulings on CRISPR-edited crops.

Yet, there is still so much to learn about this prokaryotic system. Recently, researchers at MIT and the National Center for Biotechnology Information (NCBI) have characterized a new CRISPR system that targets RNA, rather than DNA (most of the known bacterial CRISPR systems target DNA substrates).  Originally discovered in the bacteria Leptotrichia shahii, the CRISPR protein C2c2 was found to be an RNA-guided enzyme capable of cutting single-stranded RNA, providing the first instance of a CRISPR system that exclusively targets RNA. These results broaden our understanding of CRISPR systems and suggest that C2c2 can be used to develop new RNA-targeting tools (Abudayyeh, 2016). This latest CRISPR discovery has once again extended the reach of this powerful technique and holds the potential for an even wider range of applications than before.

References

Abudayyeh, O et al. C2c2 is a single-component programmable RNA-guided RNA-targeting CRISPR effector. Science 2016; 353. doi:10.1126/science.aaf5573

Begley, S. “Monsanto licenses CRISPR technology to modify crops — with key restrictions”. Stat News Sept 22, 2016. Web https://www.statnews.com/2016/09/22/monsanto-licenses-crispr/

Brodwin, E. “Monsanto may have just ended the war on GMOs”. Business Insider Sept 22, 2016. Web http://www.businessinsider.com/monsanto-ends-gmo-war-crispr-2016-9

Cyranoski, D. Chinese scientists to pioneer first human CRISPR trial. Nature News July 28, 2016; 535, 476-477.

Kaiser, J. “First proposed human test of CRISPR passes initial safety review”. Science News June 21, 2016. Web http://www.sciencemag.org/news/2016/06/human-crispr-trial-proposed

Kerr-Enskat, K. “DuPont Pioneer Announces Intentions to Commercialize First CRISPR-Cas Product”. April 18, 2016. Web https://www.pioneer.com/home/site/about/news-media/news-releases/template.CONTENT/guid.1DB8FB71-1117-9A56-E0B6-3EA6F85AAE92

Pardoll, DM. The blockade of immune checkpoints in cancer immunotherapy. Nature Reviews Cancer 2016; 12, 252-264.

U.S. Food and Drug Administration. “FDA approves Keytruda for advanced non-small-cell lung cancer”. October 2, 2015. Web http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm465444.htm

Su, S. CRISPR-Cas9 mediated efficient PD-1 disruption on human primary T cells from cancer patients. Nature Scientific Reports 2016: 6, Article 20070.