CRISPR-Cas13d: A Guideline Blueprint for Targeted RNA Therapy in Huntington's Disease

1. Abstract

​Huntington’s Disease (HD) is a genetic neurodegenerative disorder characterized by its incurable nature, highly debilitating progression, and fatality. Its symptom onset is illustrated by a variety of symptoms: depression, chorea, and suicidality. In recent years, gene therapies have reached the final phases but ultimately proved ineffective or failed, rendering the disease incurable and unpreventable. 

With the advent of CRISPR technologies, particularly CRISPR-Cas13d, there is now the possibility to further research modifying a mutated HTT allele to mitigate symptoms and slow or halt disease progression. However, the use of CRISPR technology comes with a variety of risks due to its relatively new application to human genome editing. These risks may be mitigated through recent successful animal trials using CRISPR-Cas13d, which targets RNA and prevents permanent modifications, reducing its risk for lethality. 

Many studies have shown success in muting the expression of the HTT gene through the use of CRISPR-Cas13d in similar animals with post-onset symptoms of HD. Despite the viability and real possibility of the use of CRISPR-Cas13d in human genome editing, the consideration of numerous concerns regarding the ethicality of human clinical trials is imperative. To bridge the gap between animal studies and human clinical trials regarding expanding treatment options for HD, this research paper establishes comprehensive guidelines to maintain integrity, ethicality, and responsibility on the basis of FDA and WHO gene-editing recommendations.

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2. Introduction & Literary Review 

Huntington’s disease (HD) is a prominent neurodegenerative disease in the world. HD causes nerve cells within the brain to decay over one’s lifespan; however, HD is a niche disease that affects the parts of your brain that control voluntary motor function and memory (Mayo Clinic). Although numerous medications treat symptoms of HD, all are focused on symptom management rather than on the mental, physical, and behavioral decline that HD causes. This leads to death for those who inherit HD (Mayo Clinic). To understand the severity of HD, the question of how and why the disease is so fatal must be explored. HD is caused by a trinucleotide repetition of CAG on the Huntington’s gene on chromosome four (Andhale & Shrivastava). Along with this, HD is inherited in the autosomal dominant manner, meaning that if an individual has a copy of the mutated gene then such gene will be expressed in the individual (Caron et al.). An offspring of a parent that has the expression of HD has a fifty percent chance to inherit and then potentially express the signs and symptoms associated with HD (Caron et al.). A standard amount of repeats of CAG on the Huntington’s gene is between 0-26 repeats on chromosome four. In accordance with this, the risk increases as the number of repeats goes up on this chromosome. Individuals who have less than 35 repeats of CAG are safe from expressing symptoms of HD and are at risk of producing children with the expansion of CAG that could fall in the range of HD. Those with 40 or more repeats of CAG on chromosome 4 are affected by HD and will express symptoms. It is important to note that if an individual has 60 repeats of CAG then symptoms will be expressed while the individual is a juvenile (UC Davis Health). This alludes to the notion that the figure of CAG repeats enlarges, and the symptoms will then be present earlier in life. It is important to note that Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology has been proposed as a potential treatment for HD and CRISPR has been wildly beneficial to HD research (Qin et al.). CRISPR is the newest method for editing genes. CRISPR was found through spacer DNA between viral amino acid repeats and this DNA is transcribed to RNA once the body has been infected with the corresponding infection. In doing so RNA guides a corresponding nuclease which then removes the viral DNA (The Jackson Laboratory). This method can vary based on the type of nuclease being used. 

CRISPR, found in the genomes of prokaryotic cells, these sequences are derived from DNA fragments and have been repurposed to cut DNA to be utilized as a gene-editing tool. This booming new technology has been employed in the medical world advancing and discovering new scientific studies. CRISPR editing abilities allow possible cures and treatments for thousands of fatal diseases, one being Huntington's Disease (HD). Huntington's disease occurs due to a mutation found on the fourth chromosome (HTT). When this mutation is present, individuals develop uncontrollable dance movements (Chorea), and abnormal bodily stature, alongside issues with behavior and emotion. With few viable and efficient treatments, CRISPR is the answer. While CRISPR has been applied to animal experimentation, it has yet to reach the human body with FDA approval. Working to apply this science is critical as it will suppress the repeat of HTT, and further terminate the appearance of the polyglutamate repeat. By implementing a set of extensive and effective guidelines, CRISPR will be a viable solution for Huntington's disease, saving the lives of 100,000 individuals diagnosed annually.

a. Key Issues with Current Huntington's Disease Treatment 

In prior attempts to create a treatment for Huntington’s Disease (HD) that would either suppress or delete the HTT allele, there have been very few successful ones. To start, an unsuccessful clinical trial was conducted using Antisense Oligonucleotides (ASOs) in an attempt to suppress the expression of the HTT allele (Rook et al.). ASOs are administered in dosage and work by targeting the process by which a cell makes an RNA copy from a piece of DNA (National Cancer Institute). It was found that ASOs are especially effective in treating neurodegenerative disorders. Before those trials were conducted, ASOs were approved to be used as a viable therapy option for another disease, found within the central nervous system, by the FDA. Furthermore, the fact that ASOs were a viable option to use in central nervous system diseases seems to have proven enough for the FDA to approve human clinical trials to use ASOs for Huntington’s treatment, in which the trials happened (Rook et al.). After the first 2 phases of this clinical trial, 98% of the 46 patients who enrolled in the trials said they experienced undesired side effects. These side effects of using can be characterized by off-target binding to sequences not in the HTT gene (Rook et al.). In March of 2021, when the trial was in phase III, an independent board that measured the safety of the trial determined the risks outweighed the benefits, leading them to recommend the patients stop being administered ASOs. Additionally, initial data from the trials showed signals of the trials being unsafe (Rook et al.). 

Further, one of the most normalized treatments for HD is drug medications such as tetrabenazine, risperidone, and clozapine. Though prescription medication can control symptoms such as uncontrolled movements (Chorea) and can manage further symptoms, it is not an effective, long-lasting treatment (John Hopkins Medicine). These prescriptions do not affect how these symptoms progress and the sad fact is that HD is a terminal disease. 
 

Next, CRISPR-Cas9 has been one of the most researched and implemented solutions when it comes to utilizing CRISPR for HD. Though CRISPR-Cas9 can be a more “permanent” solution to HD, there are a large variety of issues when it comes to the technology. Issues such as off-target gene modifications, cell toxicity, and runaway effects. 
 

Induced Pluripotent Stem Cells (iPSCs) is a treatment that researchers and scientists alike have tried to use to start human trials. iPSCs are a type of stem cell that scientists reprogram as a “specimen” before they start human trials. When experimenting using iPSCs to try to treat Huntington’s Disease, they use the stem cells as a substitute for human specimens. After further conclusion, the success rate for experiments using iPSCs to treat Huntington’s was between 0.1%-0.01% (Rao et al.) . 

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b. Defining CRISPR-Cas9 versus CRISPR-Cas13d

CRISPR technology often has small clusters of genes called Cas genes. There are up to 93 Cas genes; however, CRISPR-Cas9 and CRISPR-Cas13d are essential Cas genes when navigating CRISPR technology concerning Huntington’s disease (HD). CRISPR-Cas9 utilizes single guide ribonucleic acid (RNA), and once the Cas gene finds the mutated HTT gene it cuts or deletes this faulty gene. Conversely, the CRISPR-Cas13d reduces faulty HTT RNA and protein levels in the striatum (Morelli et al.). In recent studies, done on mice, significant research portrayed that CRISPR-Cas9 and the permanent suppression of the endogenous HTT gene lead to embryonic lethality and significantly depleted the HTT gene which is necessary for flourishment (Yang et al.). Further research proves that using a Cas13d-CAGEX system which is designed to edit, and silence the faulty HTT messenger mRNA in mice leads to improved motor coordination and fewer mutant HTT proteins which means decreased progression of HD. Lastly, these benefits mended several issues with CRISPR and Cas gene clusters such as the prominent off-target modifications. CRISPR-Cas13d systems prove extremely valuable, as in mice and rats the systems halted HD symptoms for eight months (Morelli et al.). Due to the fact that HD is a complex condition, finding a cure would be extremely difficult. Thus, utilizing this gene therapy to delay the progression and mitigate symptoms of HD, is revolutionary currently. 

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3. Essential Question

What is the most viable treatment for HD and what factors contribute to the challenges in transitioning from successful animal trials to human trials?

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4. Proposal: Establishing CRISPR-Cas13d: The Need for Human Trials 

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CRISPR-Cas13d systems are the most effective therapy for analyzing, mitigating, and potentially curing Huntington’s disease (HD). Not discussed earlier, pig trials have also been conducted which have shown promise in mitigating prominent symptoms of HD in pigs (Yan et al.). However, it is visible that CRISPR-Cas13d has not progressed in animal trials. To ensure that this gene-editing system is progressing past animal trials, guidelines must be placed that address consumer concerns. Currently, CRISPR-Cas13d technology is exceedingly controversial due to the lack of general knowledge of CRISPR-Cas13d and the extreme power of this technology. However, with education, strict guidelines, and rules, this gene-editing system will flourish and lead to human trials. CRISPR-Cas13d has the potential to be the most effective treatment for post-onset Huntington's Disease, provided we can overcome the challenges in translating findings from animal studies to beginning human clinical trials. To achieve this, a comprehensive set of guidelines and procedures must be established to bridge such a critical gap. 
 

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5. Rules and Guidelines for Human Trials 

Considering the advanced technology that is CRISPR-Cas13d gene-editing, this then begs the response of an all-encompassing list of guidelines that initiates and maintains responsible use of such technology. In accordance with this, Huntington’s Disease(HD) has no true rules regarding treatment surrounding HD with gene-editing. Although there have been theoretical guidelines proposed by organizations such as the Food and Drug Administration (FDA) in the United States, there still is a lack of research surrounding the use of gene therapy to treat HD. In regard to this, a list of guidelines regarding the ethical use of gene therapy to treat HD has been proposed below. 
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Table 1 - Guidelines Regarding the Use of CRISPR-Cas13d in HD For Clinical Human Trials

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Note. This is an original table created by Cooper Calis, Anshul Katta, Samadhi Liyanapathirana, Luke McFarlane, Jashna Oza, Ananya Ramesh, Aashika Reddy, and Nandini Uppala. This table uses guidelines established for using CRISPR Cas-13d in human clinical trials. It evaluates the guidelines and the motive for creating such guidelines. This data was obtained from: the U.S. Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research. (2024, January). Human Gene Therapy Products Incorporating Human Genome Editing Guidance for Industry [Review of Human Gene Therapy Products Incorporating Human Genome Editing Guidance for Industry]. https://www.fda.gov/media/156894/download

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Table 2 - Guidelines Regarding the Use of CRISPR-Cas13d in HD For Non-Clinical Human Trials

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Note. This is an original table created by Cooper Calis, Anshul Katta, Samadhi Liyanapathirana, Luke McFarlane, Jashna Oza, Ananya Ramesh, Aashika Reddy, and Nandini Uppala. This table uses guidelines established for using CRISPR Cas-13d in human non-clinical trials. It evaluates the guidelines and the motive for creating such guidelines. This data was obtained from: the U.S. Department of Health and Human Services Food and Drug Administration Center for Biologics Evaluation and Research. (2024, January). Human Gene Therapy Products Incorporating Human Genome Editing Guidance for Industry [Review of Human Gene Therapy Products Incorporating Human Genome Editing Guidance for Industry]. https://www.fda.gov/media/156894/download

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6. Limitations, Implications & Dialectic 

The following limitations, implications, and dialectic operate on the assumption of the success of CRISPR-Cas13d technology in the treatment and prevention of Huntington’s Disease in humans. Due to the rapid modernization of technology surrounding CRISPR-Cas13d, several implications and limitations arise. First, legally, preventative genome editing such as CRISPR presents concerns about the impact on future generations. Specifically, questions come to light when discussing CRISPR-Cas13d for Huntington’s Disease (HD) in-utero, on children, and pre-onset, and how this technology will affect future generations. These various age groups raise concerns due to questions regarding their voice in the decision to use CRISPR-Cas13d on the HD mutated gene. However, with technology as advanced as CRISPR-Cas13d, consensual implications will be apparent. Though, medical and everyday decisions have an impact of affecting future generations and offspring. Thus, proper measures must be taken (as specified in the list of guidelines) to ensure that proper consent measures are taken. Further, widely known, federal medical legislation could have significant leeway for private institutions due to the lack of government funding for these private practices. However, the United States government has the ability to impose legislation to ensure guidelines surrounding CRISPR-Cas13d technology and how far this technology could go. CRISPR-Cas13d could be utilized for bioterrorism and could end in fatal modification of the different genes (Lau, 2021). With the misuse of CRISPR-Cas13d technology weaponization, severe complications could occur. 
 

In fact, in 2018, Chinese scientist Dr. He Jiankui alleged that he successfully created the first genetically edited babies. Lulu and Nana’s immunity to Human Immunodeficiency Virus (HIV) and supposed cognitive improvement, while it did serve as a leap forward in the practical use of CRISPR-Cas9 human genome editing, also provoked universal ethical concerns. As a result, most human CRISPR trials were put on hold or entirely abandoned. However, this does not necessarily translate to the use of CRISPR-Cas13d in HD. The Wexelblatt Effect, as coined by Robert Wexelblatt, also raises ethical concerns regarding “unknown unknowns” and the Runaway Effect. In these regards, due to an HD CRISPR-Cas13d human clinical trial being the first of its kind, scientists cannot possibly predict all of its implications. For example, through modifying a mutant HTT allele, the genotypic to phenotypic expression of the wildtype (functioning) HTT allele may be impeded, garnering potential lethality. Due to its highly progressive, terminal, and unpreventable nature, the potentially harmful effects of HD human genome editing addressed earlier in this research paper are rendered irrelevant. 
 

Within the scope of our research, focusing on the potential for the treatment of post-onset treatment (and eventual potential prevention) of HD, most human clinical trial patients will likely be looking at end-of-life care within the next decade. So, while it is ultimately up to the patient (as stipulated in Table 1, Row 2), the potential benefits of genome editing outweigh the potential for failure. This aligns with the likely majority intuition in the HD community, as most with the illness are likely to consider the fact that the neurodegeneration of HD does not provide any significant gain of function or other tangible benefits. Additional ethical questions are raised upon the consideration of Nancy and Alice Wexler, sisters who worked at the forefront of the identification of the mutated HTT allele concerning HD. Their family history experience with HD led to questions regarding the role of pre-onset identification of the mutated HTT allele (more than 40 repeating CAG sequences) because one sister identified the presence of the nucleotide repeats in her genome, as well as the symptom onset of their mother, which meant they had a 50% chance of developing the disease. Ultimately, one sister tested her genome while the other did not, as there are no viable preventative or treatment options for the disease. This raised the question: What is the purpose of discovering one’s predisposition to HD when there is nothing that can be done about it? However, through a human clinical trial such as the one proposed in this paper, HD success can be used as an exemplar of CRISPR-Cas editing in the human genome as a means of determining whether it is unacceptable versus acceptable. 

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7. Conclusions​

With a set of guidelines and regulations in place, gene-editing methods such as Cas-13d can be utilized to alleviate the symptoms of Huntington's Disease, overall improving an individual's quality of life. These guidelines in place will ensure an effective implementation of CRISPR-13d and aid thousands of individuals globally. However, these benefits must be considered alongside additional aspects of the treatment such as ethical concerns. All in all, the application of CRISPR-13d alongside these guidelines and regulations is an effective treatment and if all practical implications are taken into consideration these frameworks can be established

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8. References.

Alkanli, S. S., Alkanli, N., Ay, A., & Albeniz, I. (2022). CRISPR/Cas9 Mediated Therapeutic Approach in Huntington’s Disease. Molecular Neurobiology, 60(3). https://doi.org/10.1007/s12035-022-03150-5 

Caron, N. S., Galen EB Wright, & Hayden, M. R. (2018, July 5). Huntington Disease. Nih.gov; University of Washington, Seattle.  https://www.ncbi.nlm.nih.gov/books/NBK1305/ 

Diversity and Inclusion in Clinical Trials. (n.d.). NIMHD. https://www.nimhd.nih.gov/resources/understanding-health-disparities/diversity-a nd-inclusion-in-clinical-trials.html#:~:text=It%20is%20essential%20to%20have

Guo, C., Ma, X., Gao, F., & Guo, Y. (2023). Off-target effects in CRISPR/Cas9 gene editing. Frontiers in Bioengineering and Biotechnology, 11(1143157). https://doi.org/10.3389/fbioe.2023.1143157 

Hildebrandt, C. C., & Marron, J. M. (2015). Justice in CRISPR/Cas9 Research and Clinical Applications. AMA Journal of Ethics, 20(9), 826–833. https://doi.org/10.1001/amajethics.2018.826. 

https://www.cancer.gov/publications/dictionaries/cancer-terms/def/transcription#. (2011, February 2). Www.cancer.gov. https://www.cancer.gov/publications/dictionaries/cancer-terms/def/transcription# Mayo Clinic. (2020, April 14). Huntington’s disease - Symptoms and causes.

Mayo Clinic; Mayo Clinic. https://www.mayoclinic.org/diseases-conditions/huntingtons-disease/symptoms-c auses/syc-20356117

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Mengstie, M. A., Azezew, M. T., Dejenie, T. A., Teshome, A. A., Admasu, F. T., Teklemariam, A. B., Mulu, A. T., Agidew, M. M., Adugna, D. G., Geremew, H., & Abebe, E. C. (2024). Recent Advancements in Reducing the Off-Target Effect of CRISPR-Cas9 Genome Editing. Biologics: Targets and Therapy, 18, 21–28. https://doi.org/10.2147/BTT.S429411

Morelli, K. H., Wu, Q., Gosztyla, M. L., Liu, H., Yao, M., Zhang, C., Chen, J., Marina, R. J., Lee, K., Jones, K. L., Huang, M. Y., Li, A., Smith-Geater, C., Thompson, L. M., Duan, W., & Yeo, G. W. (2022). An RNA-targeting CRISPR–Cas13d system alleviates disease-related phenotypes in Huntington’s disease models. Nature Neuroscience, 26(1), 1–12. https://doi.org/10.1038/s41593-022-01207-1 

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Rook, M. E., & Southwell, A. L. (2022). Antisense Oligonucleotide Therapy: From Design to the Huntington Disease Clinic. BioDrugs. https://doi.org/10.1007/s40259-022-00519-9 

Singh, A., Jasra, I., Mouhammed, O., Dadheech, N., Ray, N., & Shapiro, J. (2023). Towards Early Prediction of Human iPSC Reprogramming Success. Machine Learning for Biomedical Imaging, 2(October 2023), 390–407. 

https://doi.org/10.59275/j.melba.2023-3d9d 

The Jackson Laboratory . (2012). What is CRISPR? The Jackson Laboratory. https://www.jax.org/personalized-medicine/precision-medicine-and-you/what-is-c rispr 

UC Davis Health. (2024). The Huntington Gene - UC Davis Huntington’s Disease Center of Excellence. Health.ucdavis.edu. https://health.ucdavis.edu/huntingtons/genetic-change.html

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Videnovic, A. (2013). Treatment of Huntington Disease. Current Treatment Options in Neurology, 15(4), 424–438. https://doi.org/10.1007/s11940-013-0219-8

Yang, S., Chang, R., Yang, H., Zhao, T., Hong, Y., Kong, H. E., Sun, X., Qin, Z., Jin, P., Li, S., & Li, X.-J. (2017). CRISPR/Cas9-mediated gene editing ameliorates neurotoxicity in mouse model of Huntington’s disease. Journal of Clinical Investigation, 127(7), 2719–2724. https://doi.org/10.1172/jci92087 

Zombie apocalypse? How gene editing could be used as a weapon – and what to do about it | Brunel University London. (2021). Brunel.ac.uk. https://www.brunel.ac.uk/news-and-events/news/articles/Zombie-apocalypse-Ho w-gene-editing-could-be-used-as-a-weapon-%E2%80%93-and-what-to-do-about it#:~:text=Weaponising%20pathogens&text=But%20CRISPR%2DCas9%20could %20theoretically