The Etiology of Alzheimer’s Disease Reveals Potential Applications for Precision-Based Gene Therapy Utilizing the CRISPR/Cas9 Protocol To Counter Mechanisms of Pathogenesis

The CRISPR-Cas9 genome-editing system is a part of the adaptive immune system in archaea and bacteria to defend against invasive nucleic acids from phages and plasmids. The guide RNA (sgRNA) of the system recognizes its target sequence in the genome, and the Cas9 nuclease enzyme that can cleave DNA in this system acts as a pair of scissors to cleave the double strands of DNA [1]. Using the Cas9 enzyme (a protein involved in a biochemical reaction), the DNA-which contains the blueprints for proteins in biological processes- can be cut and edited to replace a mutated gene in an individual with a functional form [17].. Since its discovery, CRISPR-Cas9 has become the most promising platform for genome engineering in eukaryotic cells. Recently, the CRISPR-Cas9 system has triggered enormous interest in therapeutic applications, particularly for Alzheimer’s Disease. . CRISPR-Cas9 can be applied to correct  disease-causing gene mutations or can be used as a blueprint for a direct treatment approach to help establish better organismal models that more faithfully mimic human neurodegenerative diseases. This technique has already shown promise in other neurological disorders, such as Huntington’s disease [2]. There is a comparable potential utility of CRISPR/Cas9 as a treatment option for Alzheimer’s Disease by targeting specific genes including those that cause early-onset Alzheimer’s, as well as those that are significant risk factors for late-onset Alzheimer’s [3].

Alzheimer’s Disease (AD), the most common cause of dementia, is a progressive and fatal neurodegenerative disorder that primarily affects older adults. Though the definitive etiology of the disease is still to be uncovered, several characteristic features of the disease have been investigated. AD is characterized by the selective damage of brain regions responsible for cognition and memory. Clinically, patients with AD most commonly exhibit  insidiously progressive memory loss, to which other spheres of cognition are impaired over several years. In addition to memory loss, patients may also experience language difficulties and loss of executive skills, symptoms that epitomize the generalized term “dementia” [4]. In essence, AD interferes with memory, thinking, and behavior severely enough to affect a person’s work, hobbies, and social life; it is inexorably progressive and fatal within 5 to 10 years. .  In the damaged regions of an AD brain, the dysfunction and death of neurons is associated with cytoskeletal abnormalities and results in a reduction in the levels of synaptic proteins (proteins necessary for neuron function) in the regions of the brain in which these neurons terminate (the location at which the neural signal is transmitted) [6]. These pathological hallmarks are abnormal intraneuronal cytoskeletal changes, known as neurofibrillary tangles (NFTs), and extracellular protein deposits called amyloid plaques.

Amyloid plaques are aggregated misfolded proteins that accumulate in the regions between nerve cells. This beta amyloid is a protein fragment cleaved post-translationally from a larger amyloid precursor protein (APP) [9]. APP is an essential glycoprotein (protein with a sugar component) that helps neurons maintain homeostasis via synaptic (space between neurons) regulation and repair, which is the reason why neurons are specialized to produce some forms of these proteins almost exclusively. In a brain without AD, these protein fragments of APP are broken down and eliminated. In AD patients, amyloid plaques are hard, insoluble accumulations of beta amyloid proteins that clump together between the nerve cells. Beta amyloid molecules are initially found in very small strands that can dissolve in the fluid between cells, which will be washed out of the brain [10]. However, the enzyme that cleaves APP into beta amyloid relies on the recognition of an amino acid sequence that is not as precise. The electrostatic interactions between amino groups and enzyme binding and catalytic sites (where the enzyme attaches and cuts) are nonspecific, allowing the enzyme to bind to a cleavage site that is wider; this results in slightly larger strands that do not dissolve and form deposits by sticking together [7]. Genetic mutations in these cleavage proteins (gamma secretase specifically) can lead to an increased production of larger beta amyloid chains that are more prone to aggregation [11]. The presence of plaques around a neuron causes it to die, the most likely mechanism being by triggering inflammation via an immune response, allowing macrophage immune cells to kill the neuron as if it was pathogenic [9]. 

Familial AD cases are typically caused by mutations in the gene for the amyloid precursor protein (APP) or in the Presenilin genes, PSEN1 and PSEN2, whose products participate in processing APP. These genetic mutations are among some of the ripe potential targets of CRISPR/Cas9 gene therapy [9]. The CRISPR/Cas9 system was found to target and cut specific DNA sequences using only a nuclease (DNA cleaving enzyme) and RNAs to target specific DNA sequences [1]. Clearly, the potential for CRISPR/cas9 in potentially correcting these autosomal-dominant mutations is real and could be pursued. This is supported by recent studies that have analyzed the potential of correcting similar kinds of mutations using this gene editing system. For example, CRISPR/Cas9 was used to correct a presenilin (PSEN2) autosomal dominant mutation in iPSC-derived neurons (human neural cells with genetic mutations that can be collected and studied) [12]. Moreover, CRISPR/Cas9 corrected the N141I mutation that was identified via the Sanger method of DNA sequencing; this led to a normalization (no longer aggregated) of the ratio of the size of cleaved beta amyloid chains [12]. Recently, a new study reported on how CRISPR/Cas9 was used to knock out the Swedish APP mutation in patient-derived fibroblasts, leading to a 60% reduction in secreted beta-amyloid, effectively reducing plaque accumulation [13].

Neurofibrillary tangles are clusters of proteins that are commonly found in the brains of AD patients; this contributes to the symptoms of neurodegeneration (specifically memory loss and dementia) via neuronal death in areas around these clusters. NFTs are insoluble fibers found where the neurons terminate; they are constituted of the protein tau which is associated with the microtubule structure of cells [7]. The microtubule assists with cytoskeletal organization and the vital business of intracellular transport in cells [8]. NFTs form when the tau protein is misfolded due to electrostatic interactions with the negatively charged phosphate group and the other domains of the protein [7]. NFTs form inside of neurons and interfere with the cellular machinery used to create and traffic proteins, eventually becoming fatal to the neuronal cell [9]. In humans, tau is expressed as both three-repeat (3R) or four-repeat (4R) isoforms (versions of the protein sequence that include repeated amino acids) due to the arrangement of the microtubule-associated protein tau gene (MAPT). Mutations in MAPT were found to cause a subtype of frontotemporal lobar degeneration (FTLD), demonstrating that this pathology is sufficient for neurotoxicity and dementia, common to AD [5].Based on these genetic findings, multiple transgenic mouse (containing human genes that were implanted into the mouse genome) lines have been created to overexpress human 4R tau containing the FTLD mutations. CRISPR/Cas9 gene editing offers a potential therapy for this mutation by reducing the overexpression of these FTLD mutations, which could reduce the risk and progression of dementia in AD patients. 

Another potential CRISPR/Cas9 gene target includes mutations involved in Apolipoprotein E (apoE); a major risk factor for developing late-onset AD is harboring the Apolipoprotein E4 (APOE4) allele [14]. Human apoE is polymorphic with three major protein isoforms, apoE2, apoE3, and apoE4, all of which differ by single amino acid substitutions in their sequence. In each one a cysteine amino acid is replaced with an arginine at amino positions 112 and 158 in the protein [15]. The E2 allele is the rarest form of APOE and carrying even one copy appears to reduce the risk of developing AD by up to 40%; E3 has no effect on AD pathology. However, having even one copy of the E4 allele increases the chances of AD from 10-15% [15]. Therefore, one potential use of the CRISPR/Cas9 system could be to convert APOE4 to APOE2 or E3. In this regard structural and functional changes from apoE4 to apoE3 or apoE2 mediated through CRISPR/Cas9 may be a viable approach to treat AD patients carrying APOE4 [16].

The potential applications of CRISPR/Cas9 are far reaching including being able to precisely and efficiently target, edit, modify, regulate, and mark genomic loci of a wide array of cells and organisms. Its application in genome-wide studies will enable large-scale screening for drug targets and other phenotypes and will facilitate the generation of engineered animal models that will benefit the understanding of human diseases [2]. The ultimate goal of this system would be to correct mutations at precise locations in the human genome in order to treat genetic causes of disease including certain neurodegenerative disorders [3]. 

This promising gene-editing tool, CRISPR/Cas9, has in the last decade become known as a novel protocol for not only generating specific neurodegenerative disease animal models for investigating the mechanisms and screening potential drugs, but also for the editing and fixing sequence-specific genes to help patients to restore protein products of these nonfunctional gene copies. The etiologies of AD, has not been completely defined, but certain potential therapeutic gene targets have been identified through research as viable approaches to preventative treatment. The sheer versatility of these gene therapy treatments for AD puts CRISPR/Cas9 at the vanguard of the precision-medicine research movement. 

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References 

1. Jones, E. L., Kalaria, R. N., Sharp, S. I., O’Brien, J. T., Francis, P. T., & Ballard, C. G. (2011). Genetic associations of autopsy-confirmed vascular dementia subtypes. Dementia and geriatric cognitive disorders, 31(4), 247–253. https://doi.org/10.1159/000327171
2. Jun Wan Shin, Kyung-Hee Kim, Michael J. Chao, Ranjit S. Atwal, Tammy Gillis, Marcy E. MacDonald, James F. Gusella, Jong-Min Lee, Permanent inactivation of Huntington’s disease mutation by personalized allele-specific CRISPR/Cas9, Human Molecular Genetics, Volume 25, Issue 20, 15 October 2016, Pages 4566–4576, https://doi.org/10.1093/hmg/ddw286
3. Barman, N. C., Khan, N. M., Islam, M., Nain, Z., Roy, R. K., Haque, A., & Barman, S. K. (2020). CRISPR-Cas9: A Promising Genome Editing Therapeutic Tool for Alzheimer’s Disease-A Narrative Review. Neurology and therapy, 9(2), 419–434. https://doi.org/10.1007/s40120-020-00218-z
4. Terry, R. D., Masliah, E., Salmon, D. P., Butters, N., DeTeresa, R., Hill, R., Hansen, L. A., & Katzman, R. (1991). Physical basis of cognitive alterations in Alzheimer’s disease: synapse loss is the major correlate of cognitive impairment. Annals of neurology, 30(4), 572–580. https://doi.org/10.1002/ana.410300410
5. Bu G. (2009). Apolipoprotein E and its receptors in Alzheimer’s disease: pathways, pathogenesis and therapy. Nature reviews. Neuroscience, 10(5), 333–344. https://doi.org/10.1038/nrn2620
6. Mirra, S. S., Heyman, A., McKeel, D., Sumi, S. M., Crain, B. J., Brownlee, L. M., Vogel, F. S., Hughes, J. P., van Belle, G., & Berg, L. (1991). The Consortium to Establish a Registry for Alzheimer’s Disease (CERAD). Part II. Standardization of the neuropathologic assessment of Alzheimer’s disease. Neurology, 41(4), 479–486. https://doi.org/10.1212/wnl.41.4.479
7. Williams D. R. (2006). Tauopathies: classification and clinical update on neurodegenerative diseases associated with microtubule-associated protein tau. Internal medicine journal, 36(10), 652–660. https://doi.org/10.1111/j.1445-5994.2006.01153.
8. Brouhard, G. J., & Rice, L. M. (2018). Microtubule dynamics: an interplay of biochemistry and mechanics. Nature reviews. Molecular cell biology, 19(7), 451–463. https://doi.org/10.1038/s41580-018-0009-y
9. De Strooper, B., & Karran, E. (2016). The Cellular Phase of Alzheimer’s Disease. Cell, 164(4), 603–615. https://doi.org/10.1016/j.cell.2015.12.056
10. Huang, Y., & Mucke, L. (2012). Alzheimer mechanisms and therapeutic strategies. Cell, 148(6), 1204–1222. https://doi.org/10.1016/j.cell.2012.02.040
11. Annaert, W., & De Strooper, B. (2002). A cell biological perspective on Alzheimer’s disease. Annual review of cell and developmental biology. https://doi.org/10.1146/annurev.cellbio.18.020402.142302
12. Dolmetsch, R., & Geschwind, D. H. (2011). The human brain in a dish: the promise of iPSC-derived neurons. Cell, 145(6), 831–834. https://doi.org/10.1016/j.cell.2011.05.034
13. György, B., Lööv, C., Zaborowski, M. P., Takeda, S., Kleinstiver, B. P., Commins, C., Kastanenka, K., Mu, D., Volak, A., Giedraitis, V., Lannfelt, L., Maguire, C. A., Joung, J. K., Hyman, B. T., Breakefield, X. O., & Ingelsson, M. (2018). CRISPR/Cas9 Mediated Disruption of the Swedish APP Allele as a Therapeutic Approach for Early-Onset Alzheimer’s Disease. Molecular therapy. Nucleic acids, 11, 429–440. https://doi.org/10.1016/j.omtn.2018.03.007
14. Weisgraber, K. H., Rall, S. C., Jr, & Mahley, R. W. (1981). Human E apoprotein heterogeneity. Cysteine-arginine interchanges in the amino acid sequence of the apo-E isoforms. The Journal of biological chemistry. https://pubmed.ncbi.nlm.nih.gov/7263700/
15. Farrer, L. A., Cupples, L. A., Haines, J. L., Hyman, B., Kukull, W. A., Mayeux, R., Myers, R. H., Pericak-Vance, M. A., Risch, N., & van Duijn, C. M. (1997). Effects of age, sex, and ethnicity on the association between apolipoprotein E genotype and Alzheimer disease. A meta-analysis. APOE and Alzheimer Disease Meta Analysis Consortium. https://pubmed.ncbi.nlm.nih.gov/9343467/
16. Dong, L. M., & Weisgraber, K. H. (1996). Human apolipoprotein E4 domain interaction. Arginine 61 and glutamic acid 255 interact to direct the preference for very low density lipoproteins. The Journal of biological chemistry, 271(32), 19053–19057. https://doi.org/10.1074/jbc.271.32.19053
17. Wu, S. S., Li, Q. C., Yin, C. Q., Xue, W., & Song, C. Q. (2020). Advances in CRISPR/Cas-based Gene Therapy in Human Genetic Diseases. Theranostics, 10(10), 4374–4382. https://doi.org/10.7150/thno.43360