Newly discovered gene editing tool CRISPR-Cas allows editing of our genome at precise locations. The tool could be used directly in the patient to cut out and repair mutations causing genetic diseases.

 

Around 10,000 human diseases are caused by an alteration of DNA, the blueprint of our bodies.1 A novel approach to treat genetic diseases uses a newly discovered gene editing tool, named CRISPR-Cas, which is revolutionizing medicine. Originally, it is a bacterial defense mechanism against foreign, invading DNA. In 2012, however, Jennifer Doudna and Emmanuelle Charpentier discovered that CRISPR-Cas can be modified to be used as a tool for precise gene editing in any organism. It works like a pair of scissors, that can cut our genome at precise locations. This triggers a DNA repair process, which in combination with insertion or modification of specific DNA sequences, can lead to precise and permanent editing of the gene.. This discovery was a major milestone in the field of life sciences and resulted in Doudna and Charpentier winning the 2020 Nobel prize in chemistry. However, another step has to be taken before the gene editing tool can work in humans. The associated components need to be brought to and enter the diseased cells to perform the cut and repair. Scientists are looking into ways to secure that the CRISPR-Cas components are present in the cells. An approach to delivery of the components uses delivery vehicles. It is due to this development that CRISPR-Cas has the potential to revolutionize gene therapy, it has therefore received extensive media attention. For example, a documentary on CRISPR-Cas, called Human Nature, was released in 2019. To quote the New York Times: “every ‘oh wow’ in Human Nature is matched by an ‘oh no’ somewhere down the line”. This statement summarizes the general attitude towards the gene editing tool quite well.2

 

Cut-and-Edit as a way to treat genetic diseases

CRISPR-Cas can be described as a kind of molecular scissors or scalpel that can specifically cut out mutations within DNA and edit them into the correct version.3 For example, it may one day be possible to use CRISPR-Cas to edit DNA by having it manipulate mutations linked to certain diseases. A navigator molecule, called guide-RNA, would guide the editing tool, named Cas, to the mutated site in the DNA.4 At the targeted site it would cut-and-edit the faulty DNA. This tool can be easily adjusted to target most regions within the genome. Therefore, it has the potential to treat a whole range of mutated regions that are causing diseases. However, there are still significant hurdles to overcome before this scalpel-molecule can be used in real life patients. For example, it remains a question whether the gene editing with CRISPR-Cas should take place within or outside of the body. Researchers are looking into ways to do the latter, which is already being done successfully in clinical trials to treat blood cancer.5 A different approach would be for CRISPR-Cas to perform its editing directly within the human body. For this approach, the tool needs to be delivered to the mutated cells in a safe and efficient manner.6

Schematic representation of gene editing with the gene editing tool CRISPR/Cas.

 
The personalized delivery shuttle of CRISPR-Cas

An approach to the delivery of Cas and guide-RNA that is explored at Utrecht University’s pharmaceutics department, is to employ small, virus-sized delivery vehicles. They carry the tools to the diseased organ, where it can correct the genetic mutation that is causing the disease. These tiny spherical vehicles are called nanoparticles. They are made of biological molecules that are not necessarily foreign to the body, as these molecules can also be found in the cellular membrane.7 It has been shown that nanoparticles can safely and efficiently deliver drug molecules.8 They protect the molecules they are carrying from molecules within the body and, simultaneously, shield the body from their cargo to prevent unwanted side effects. The idea is that the nanoparticles that carry the Cas and guide-RNA will be injected into the bloodstream with a syringe, where they circulate until they reach the desired destination. For example, if the liver was the diseased organ, the delivery would be assured by modifying the nanoparticles’ outer surface with specific molecular components that bind receptors on liver cells.9 The nanoparticles will then stick to the liver cells when passing by and thereafter be absorbed by the liver cells.

 
From a lab bench to the patient

The aim is to develop and improve the delivery vehicles that carry Cas and guide-RNA to diseased cells and perform gene editing and repair. First results show that nanoparticles can be constructed to carry the gene editing tool. Cas and guide-RNA are sensitive molecules, though. They can be damaged in the process of making nanoparticles, resulting in loss of function. The experiments performed in the labs study the ideal physiological conditions in which the gene editing tool stays viable.10 The next step is to observe whether the nanoparticles can enter cells and release Cas and guide-RNA effectively by using microscopy. These experiments can also help investigate and mitigate any toxicity caused by the nanoparticles and gene editing tool. Another challenge in using the gene editing tool in humans is to prevent the immune system from clearing CRISPR-Cas out of the body. Since CRISPR-Cas is foreign to the human body and originates from bacteria, the immune system may interfere with treatment efficiency. Using nanoparticles for delivery already addresses this concern, however, only up to the moment the gene editing tool is released into the cell. Studies at the Pharmaceutics department are also dedicated to developing a solution to avoid an immune response against the gene editing components. Once this problem is addressed, the developmental work will be performed in mice first. This is done because at this early stage in the developmental process, undesired and serious adverse effects might still occur, which first need to be addressed adequately before moving on to human trials. For example, one major concern is that editing may occur at a different DNA-site than intended, leading to unwanted side-effects. In the case of faulty editing, the gene editing components and delivery vehicle should be altered in the laboratories, until the most precise application of the tool for gene therapy is found.

 

Are we messing too much with nature?

The current research focuses on showing that the delivery vehicles can carry Cas and guide-RNA to the diseased cell and perform editing and repair there. However, in order to use the potential of CRISPR-Cas in gene therapy, its working mechanisms need to be better understood. Critical voices suggest that gene therapy might be too much of an interference with nature. Are permanent changes to our DNA, the blueprint of our bodies, not too risky? I believe that it is important for scientists in this field to keep this concern in mind when performing their research. Scientists have agreed, for example, not to modify the germ line, meaning the cells involved in reproduction. That means that any changes would affect the individual only and are not passed on to the next generation. CRISPR-Cas gene editing research is therefore only focused on correcting mutations in non-reproductive cells, the so-called somatic mutations.11 Addressing moral concerns within the experiments can only contribute to an increased understanding of and control over CRISPR-Cas. In cases where the usage of the gene editing tool would be too risky, scientists should report their findings and reconsider the application of CRISPR-Cas in gene therapy. I believe the ability of CRISPR-Cas to definitively cut out the error causing a disease, outweighs the arguments against further research into gene therapy. I hope the experiments will show the promising future of CRISPR-Cas and help settle public concerns. The translation of CRISPR-Cas as a technique to a usable treatment is still in its early stages either way. The ongoing research in many labs worldwide and in the Pharmaceutics department of Utrecht University are only the first steps in making the technology widely available. Generally, new therapeutics take up to 15 years before they enter the market. Nonetheless, I am very excited to be a part of the process of developing novel approaches to curing diseases.

 

References

1 sd. WHO. accessed October 19, 2020, https://www.who.int/genomics/public/geneticdiseases/en/index2.html.

2 Jaworowski, K., The New York Times. 12 March 2020, accessed October 30, 2020. https://www.nytimes.com/2020/03/12/movies/human-nature-review.html.

3 Huai, Cong, Gan Li, Ruijie Yao, Yingyi Zhang, Mi Cao, Liangliang Kong, Chenqiang Jia, et al. “Structural Insights into DNA Cleavage Activation of CRISPR-Cas9 System.” Nature Communications 8, no. 1 (2017): 1–9.

4 Lim, Youngbin, So Young Bak, Keewon Sung, Euihwan Jeong, Seung Hwan Lee, Jin Soo Kim, Sangsu Bae, and Seong Keun Kim. “Structural Roles of Guide RNAs in the Nuclease Activity of Cas9 Endonuclease.” Nature Communications 7 (2016): 1–8.

5 Haridy, R., “More early data revealed from landmark CRISPR gene editing human trial.” New Atlas (2020).

6 Wilbie, D., Walther, J., and Mastrobattista, E., “Delivery Aspects of CRISPR/Cas for in Vivo Genome Editing”, Accounts of Chemical Research, 2019.

7 Xu, C. F. et al., “Rational Designs of in Vivo CRISPR-Cas Delivery Systems.” Advanced Drug Delivery Reviews, 2019.

8 Akinc, A. et al., “The Onpattro Story and the Clinical Translation of Nanomedicines Containing Nucleic Acid-Based Drugs.” Nature Nanotechnology 14, no. 12 (2019), 1084–1087.

9 Jing, F., Li, J., Liu, D., Wang, C., and Sui, Z., “Dual Ligands Modified Double Targeted Nano-System for Liver Targeted Gene Delivery.” Pharmaceutical Biology 51, no. 5 (2013), 643–49.

10 Wei, T., Cheng, Q., Min, Y. L., Olson, E. N., and Siegwart. D. J., “Systemic Nanoparticle Delivery of CRISPR-Cas9 Ribonucleoproteins for Effective Tissue Specific Genome Editing.” Nature Communications 11, no. 1 (2020), 1–12.

11 On, Roup. “European Group on Ethics in Science and New Technologies (EGE) Statement on Gene Editing.” Jahrbuch Für Wissenschaft Und Ethik 21, no. 1 (2017).

 

Images

Header: DNA by Thom Hamer.

Image 1: Schematic Representation of Gene Editing with the gene editing tool CRISPR/Cas. Created with BioRender.com.

Editor: Stephanie Bosschaert

Johanna Walther

Johanna Walther (1993) obtained her bachelor’s degree in biochemistry at the Martin-Luther University in Halle (Saale), Germany in 2016. Afterwards, she moved to Utrecht, the Netherlands for a gap year as a junior scientist at the pharmaceutical start-up company Cristal Therapeutics in collaboration with the Department of Pharmaceutics, Utrecht University. In 2017, Johanna started the master’s program Drug Innovation at Utrecht University, during which she performed a major research internship at the Department of Pharmaceutics on delivery vehicles for CRISPR/Cas for gene therapy. With this thesis she won the Dr. Saal Van Zwanenberg Onderzoeksprijs in 2019. After obtaining her Masters in Science, Johanna started as a PhD candidate back at the Department of Pharmaceutics under supervision of prof. Enrico Mastrobattista on the design of a delivery vehicle for CRISPR/Cas for gene therapy.