How can we incorporate Crispr Gene-Editing Technology to prevent prion diseases such as CJD?

By Neelesh Sathish

Credit: istockphoto.com

Specific Aims:

There is a growing concern of prion diseases which are fatal neurodegenerative diseases with no available treatments as of right now. The formation of prion diseases occurs from the misfolding of the cellular prion protein with no reason as to why. Current approaches have failed due to the relatively low information gathered upon it in research. Current approaches have also tried to deplete prion proteins within the brain but have failed due to the importance of these proteins within the brain. This project aims to utilize Crispr Gene-Editing technology to prevent the formation of prion diseases through preventing the cellular prion proteins from misfolding. If this project were to be successful, it would revolutionize the treatment and prevention of prion diseases, and provide a novel solution to a severely detrimental problem. 

The first specific aim of this project is to present the importance of the prion proteins within our brain. During an experiment, scientists removed prion proteins from chimeric mice and found that mice that lacked prion proteins suffered from extensive oxidation damage, unlike mice with prion proteins, showcasing the benefit prion proteins produce for the body. Also, neurons with prion proteins are significantly more resistant to copper toxicity than cells that lack prion proteins. The advantages prion proteins provide for the body showcases the necessity of a technology that doesn’t deplete prion proteins, but prevents misfolding.

The second specific aim of this project is to edit the gene that provides instructions for the formation of cellular prion proteins (PRNP gene). I hypothesize that through precise modifications to the PRNP gene, we can prevent normal cellular prion proteins (PrPC) from misfolding into the fatally infectious, misfolded form (PrPSc). We would need to create a ‘guide’ RNA specific to the PRNP gene and utilize Crispr Gene-Editing technology to induce targeted modifications. To test if the solution has worked, we would expose the edited PrPC to PrPSc and observe if they resist misfolding. We would use Western blotting and other techniques for evaluating the results.

The third specific aim of this project is to prevent prion disease formation in vivo using Crispr Gene-Editing. I hypothesize that the utilization of Crispr Gene-Editing within mice will prevent the development of prion diseases. We will use chimeric mice, which are genetically engineered mice, that produce PrPC and are susceptible to prion diseases. We will employ Crispr Gene-Editing technology to edit the PRNP gene within the mice. We will observe the mice through behavioral and molecular analyses which will validate the findings. We will also observe the mice to look for any side effects that may have formed from the genetic modifications. 

Background/Significance:

Prion diseases include various neurodegenerative diseases such as Creutzfeldt-Jakob disease in humans, scrapie in sheep, and bovine spongiform encephalopathy ("mad cow disease") [1]. The main cause of prion diseases is the misfolding of the prion protein (PrP), converting it from its normal cellular form (PrPC) into a fatally infectious, misfolded form (PrPSc) [2]. Scientists do not understand why the PrPC misfolds into the PrPSc. Prion diseases are linked with memory loss, behavior changes, and movement problems in the body [3]. Each year, an average of 247 people die from prion diseases and over 300 cases are reported [8] [9]. One the current treatments for prion diseases include PrPC knockdown where the amount of PrPC in the body is reduced [4]. Although this could work, it would pose major disastrous effects to the body. This is due to the fact that prion proteins actually play a major function in our brain. Dr. Zhao-Yun Wang et al. removed PrP from chimeric mice and found that mice that didn’t have PrP suffered from detrimental oxidation damage, which was not the same with mice that had PrP, presenting the benefit that prion proteins produce for the body. As well as this, neurons that have PrP are much more resistant to copper toxicity than neurons that don’t have PrP [5]. Instead of reducing the amount of PrPC in the brain, it would be possible to utilize a technology known as Crispr Gene-Editing to essentially prevent PrPC from turning into PrPSc. Crispr Gene-Editing essentially is a natural process that scientists harnessed in order to be able to alter DNA and change specific genes in an organism [6]. 

In the process of Crispr Gene-Editing, first a ‘guide’ RNA is designed to match the gene that needs to be edited. RNA stands for ribonucleic acid which is present in all living organisms just like DNA. The ‘guide’ RNA is then attached to Cas9, which is an enzyme that plays the role of ‘molecular scissors’ as it can cut DNA at a specific location so that the DNA can be altered. After this, the ‘guide’ RNA guides Cas9 to the intended gene and the Cas9 cuts through the DNA [7]. From there scientists can induce a template DNA that will cause the intended DNA to rebuild with a function that scientists intended for. In this case if the DNA of prion proteins could be edited to prevent it from misfolding into PrPSc, it would mean that prion diseases wouldn’t occur and would be highly rewarding.

The Crispr Gene-Editing technology isn’t the only technology that is able to conduct gene-editing. Other technologies such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) also have the ability to gene-edit with much precision. The reason why Crispr Gene-Editing technology would be much better for this project is the fact that this technology uses a different approach than other technologies which makes it able produce high precision and accuracy in targeting DNA sequences. As well as this, Crispr Gene-Editing technology is much more rapid, has a relatively low cost, and is highly flexible [10]. 

Future Directions:

To observe how the Crispr Gene-Editing technology effectively prevents prion diseases like CJD, we can look at three interrelated future directions. Each direction will provide a specific idea and a specific way to test the idea. These directions together will showcase the way Crispr Gene-Editing technology can prevent prion diseases. 

My hypothesis for the first future direction is that the role of prion proteins in the brain is highly important and if the prion proteins were to be depleted then it would produce detrimental effects. We can test this in vivo through the utilization of chimeric mice. We can first set a control group with mice that will have prion proteins within their brain and we can then set up an experimental group that will be depleted of prion proteins within the brain. We can observe the mice to see how the behavior changed in the experimental group versus the control group. Along with this we can look at the health of the mice within the experimental group to observe whether it is better or worse than the control group. This would allow us to understand if prion proteins are even vital for our bodies. 

My hypothesis for the second future direction is that changing the PRNP gene to a gene that doesn’t allow PrPC to misfold into PrPSc using Crispr Gene-Editing would prevent prion diseases from forming. When designing the ‘guide’ RNA, we would need to match it to be exactly the same as the PRNP gene so that the ‘guide’ RNA would be able to direct the Cas9 protein to the intended location. After this we would need to utilize Crispr Gene-Editing technology to make changes within the PRNP gene to make it so that the PrPC doesn’t misfold into the PrPSc. To test if this editing and matching works we would expose the edited cells to PrPSc to see if any of the cells misfold (as when PrPSc comes in contact with PrPC the PrPC would misfold into PrPSC as well like a zombie). We would then look through Western blotting and other techniques to see if the editing makes it so that PrPSc doesn’t form. This would allow us to understand how Crispr Gene-Editing technology will deplete the chance of the formation of PrPSc.

My hypothesis for the third future direction is that utilizing Crispr Gene-Editing technology in the PRNP gene in living animals will stop prion diseases from developing. We would first utilize genetically engineered mice that are capable of producing PrPC. We would need to make sure these mice are susceptible to prion diseases. Then we would induce Crispr Gene-Editing technology into the mice and change the PRNP gene. We would then see if there is any sort of sign in the mice that prion diseases are developing or not. We would also see if there are any side effects within the mice. This would allow us to understand how Crispr Gene-Editing technology would work in vivo. This could help us with future administration of Crispr within humans instead of just animals. 

Works Cited:

Linden, R., Martins, V. R., Prado, M. A., Cammarota, M., Izquierdo, I., & Brentani, R. R. (2008). Physiology of the prion protein. Physiological Reviews, 88(2), 673–728. https://doi.org/10.1152/physrev.00007.2007

Prusiner, S. B., Scott, M. R., DeArmond, S. J., & Cohen, F. E. (1998). Prion protein biology. Cell, 93(3), 337–348. https://doi.org/10.1016/s0092-8674(00)81163-0

Seladi-Schulman, J. (2023, April 10). Prion disease: Symptoms, causes, treatment, & prevention. Healthline. https://www.healthline.com/health/neurological-health/prion-disease

Kong, Q. (2006). RNAi: A novel strategy for the treatment of prion diseases. Journal of Clinical Investigation, 116(12), 3101–3103. https://doi.org/10.1172/jci30663

Dong, X.-P. (2011). Knockdown of prion protein (PRP) by RNA interference weakens the protective activity of wild-type PRP against copper ion and antagonizes the cytotoxicity of fcjd-associated PRP mutants in cultured cells. International Journal of Molecular Medicine. https://doi.org/10.3892/ijmm.2011.688

=. (2023, April 25). The Basics of CRISPR Gene Editing. Cleveland Clinic. https://health.clevelandclinic.org/crispr-gene-editing

What is CRISPR-Cas9?. Your Genome. (n.d.). https://www.yourgenome.org/theme/what-is-crispr-cas9/#:~:text=The%20CRISPR%2DCas9%20system%20consists,then%20be%20added%20or%20removed

Holman, R. C., Belay, E. D., Christensen, K. Y., Maddox, R. A., Minino, A. M., Folkema, A. M., Haberling, D. L., Hammett, T. A., Kochanek, K. D., Sejvar, J. J., & Schonberger, L. B. (2010). Human prion diseases in the United States. PLoS ONE, 5(1). https://doi.org/10.1371/journal.pone.0008521

Prion Diseases. Johns Hopkins Medicine. (n.d.). https://www.hopkinsmedicine.org/health/conditions-and-diseases/prion-diseases

How does CRISPR compare with other gene-editing methods? | biocompare.com. (n.d.). https://www.biocompare.com/Editorial-Articles/576583-How-Does-CRISPR-Compare-with-Other-Gene-Editing-Methods/