Novel g-RNA CRISPR Nanocapsules targeting HSV-1 in Alzheimer’s disease as a model for therapeutic development

Abstract

It is known that there exists a correlation where herpesvirus acts as a transducer in

biological pathways that stimulate Alzheimer’s.. Once contracted, herpes virus can remain

dormant and travel to the brain. There, it activates natural cellular responses that, due to viral

intervention, become harmful biochemical pathways. Such responses include the endoplasmic

reticulum stress, which is a natural response to irregular protein folding, and results in

amyloid-beta accumulation, the leading factor in Alzheimer’s Disease. With the increasing

prevalence of CRISPR, and in conjunction with the Cas-9 protein, such a system may be

employed to deliver gene-editing proteins in the brain to edit both human and viral genomes.

This would require a specifically-engineered gRNA that can precisely edit the genome of the

HSV-1 virus along with a delivery system that has the ability to bypass the blood-brain barrier

and induce endocytosis within microglia. This approach will reduce the reproduction rate and

prevent activation of Alzheimer’s associated pathways.

Introduction

Alzheimer’s disease is a neurodegenerative disorder that hinders and destroys the

cognitive functions of humans. It affects more than 44 million people worldwide, with patients

typically diagnosed at an age of 651. Studies have shown that there is a significant link between

the herpes simplex-1 virus and the development of Alzheimer’s disease, with some studies

seeing over 50 percent of Alzheimer’s patients having HSV-1 in their brains2. HSV-1 is a virus

that once contracted, can remain latent in the nervous system. Under conditions such as stress,

immune suppression and aging, HSV-1 can reactivate and travel to the brain. When activated in

the brain, it triggers a reproduction phase that infects brain microglial and astrocyte cells.

Background

One consequence of this reproduction is the release of viral proteins in the cells through

the endoplasmic reticulum (ER). The production of viral proteins during HSV-1 replication

overwhelms the ER, which triggers a natural cellular response called the unfolded protein

response (UPR). The purpose of UPR is to restore the normal ER functions, but this process

activates transcription factors that alter important biochemical pathways within the brain. One

notable effect is the upregulation of the BACE-1 enzyme3. BACE-1 is a secretase responsible for

the cleavage of the amyloid precursor protein (APP) into smaller fragments. The fragments

produced include amyloid-beta, the primary component of amyloid plaques, which is responsible

for the pathology of Alzheimer’s disease.

So far, there is no substantial solution to address the correlation between HSV-1 and

Alzheimer’s disease. Current attempts at a solution include antiviral therapies. Medications like

acyclovir, valacyclovir, and famciclovir target the replication of HSV-1. They inhibit DNA

polymerase within the virus in an attempt to hinder the reproductive functions of the virus4.

While they are effective in treating active HSV-1, the latent versions of the herpesvirus are

unaffected by these drugs. Additionally, these medications are not effective in combating the

virus when it is active within the brain.

However, gene-editing technologies, such as CRISPR, present promising methods worth

investigating. CRISPR-Cas9 is a genome-editing technology that allows for the modification of

DNA with precision. A customized gRNA with a desired sequence binds to the Cas9 protein, and

the resulting complex scans the DNA strand to find the sequence specified by the gRNA. The

Cas9 protein then creates a double-strand break in the DNA through two nuclease domains

(RuvC and HNH).

In February of last year, an important advancement in CRISPR was achieved. A special

delivery system was developed to allow the transport of CRISPR components over the

blood-brain barrier. Nanocapsules conceal the components and attach to the epithelial cells’

receptors lining the blood brain barrier. It induces endocytosis and allows passage of CRISPR.

This innovation is significant towards our own methodology5.

Future Technology

In the near future, gene-editing technology could reach a height where CRISPR-Cas9

revolutionizes how we approach HSV-1 and its pathology in Alzheimer’s. We can change the

genome of our human cells or simply edit the genome of the herpes virus to remove the features

that facilitate the harmful biochemical pathways. One possibility is to remove the genes that code

for the surface receptor proteins on the cells through CRISPR.

During reproduction, HSV-1 attaches to the surface proteins of cells like microglia and

astrocytes. By attaching to these surface proteins, it gives the virus a way into the cell to activate

its reproductive functions through the endoplasmic reticulum. The cell receptors responsible are

nectin-1 and heparan sulfate6. HSV-1 uses viral glycoproteins (gB, gH, gL, and gD) that are

essential for attaching to these cellular receptors. If CRISPR-Cas9 cleaves the portions of DNA

that codes for these proteins, it would effectively block the biochemical pathway that induces

Alzheimer’s disease. Similar to the cell receptors, it would need a design for a gRNA strand that

matches the sequence for the protein7. If the correct sequence can be derived and successfully

applied, there is potential for clinical treatment for patients with herpes. By altering the genes

using CRISPR-Cas9, it would suffice in reducing the risk of developing Alzheimer’s disease.

Not only does it prevent the initiation of harmful biochemical pathways, it offers a solution to

mitigate the progression of existing disease by diminishing viral activity. This approach has the

potential for providing a preventative and therapeutic breakthrough for millions worldwide.

Methods

Despite the promising results of CRISPR-Cas9 technology to edit the genome, the

technology comes with numerous difficulties that need significant scientific breakthroughs to

overcome. One difficulty is the transport of CRISPR-Cas9 components across a natural barrier.

The blood-brain barrier is a semi-permeable membrane made of endothelial cells within

capillaries leading to the brain8. It plays a central role in homeostasis and protecting the brain

from pathogens. Even though HSV-1 is able to bypass the barrier, the tight junctions created by

the epithelial cells prevent the passing of crucial components of CRISPR-Cas9. There must be a

delivery system that can transport the components to precise locations within the brain. This

presents a need for a breakthrough that satisfies these requirements.

However, in April of 2022, researchers engineered a strategy to deliver CRISPR-Cas9 to

brain tumors that is both efficient and safe. Using nanocapsules specifically designed for

glioblastoma cells, the researchers succeeded in reaching a “gene editing efficiency in a brain

tumor (up to 38.1%)”9, and “a negligible (less than 0.5%) off-target gene editing in high-risk

tissues” (same source). Despite this ground-breaking delivery system, it is engineered to target

glioblastoma cells only. It would be necessary to adapt this system to selectively target glial cells

that are infected by HSV-1. Modifications to the targeting ligands would be required to ensure

specificity and avoid off-target effects.

A study can be conducted to identify and validate the ligand capable of binding to HSV-1

infected cells. Similar methodology compared to the glioblastoma cell delivery system can be

applied. The experiment will go through multiple phases:

1. Cell Preparation and Infection

Glial cells such as astrocytes and microglia will be cultured and induced HSV-1 infection.

HSV-1 infection can be confirmed using techniques such as qPCR for viral markers. Western

blot will be crucial for protein expression analysis for stress-related proteins.

2. Ligand Selection

We can utilize a phage display peptide library to identify peptides that bind to the surface

of HSV-1-infected glial cells. Phages will be incubated with infected cells. Additional binders

that are non-specific to the cells will be removed through washing steps. Then, bound phages

will be eluted and amplified for further rounds of selection, with increased specificity applied in

each round for high-affinity binders10.

3. Functionalization of Nanocapsules

The ligands will be conjugated to nanocapsules for the delivery system. The

nanocapsules can be tracked with fluorescent dyes or CRISPR-Cas9 components to evaluate

their targeting and uptake efficiency in vitro.

4. In Vivo Evaluation

Finished nanocapsule-ligand apparatuses will be tested in a mouse model infected with

HSV-1 to evaluate their ability to cross the blood-brain barrier. Nanocapsule targeting specificity

can be assessed through fluorescence imaging.

Rejected Alternatives

One solution considered was to develop a treatment that strengthens the filtering ability

of the blood-brain barrier in order to prevent the passing of HSV-1 virus completely. Possibilities

of this would involve targeting the epithelial cells that line the blood-brain barrier. By increasing

the functionality of their tight junctions, it would effectively block the pathogens from crossing

the barrier. While completely preventing the virus from entering the brain would be an ideal

solution, it would not address the problem that latent HSV-1 already exists within the nervous

system. Through neuronal pathways, the virus can still reactivate and travel to the brain. This

would render the solution completely useless11. Additionally, modifying the blood-brain barrier

may bring issues that disrupt its natural biological functions. The blood-brain barrier serves as a

wall that regulates the passage of particles, letting across nutrients essential to the health of the

brain. Current technology would not be able to provide selective control, making it difficult to

prevent viral passage without disrupting normal brain function. The CRISPR-Cas9 approach is

more optimal because rather than altering a natural physiological barrier, it would target infected

cells.

Another solution considered was to change the genome of the cell receptors on the cells

that HSV-1 tends to infect within the brain. The receptors, nectin-1 and heparan sulfate, would be

altered using CRISPR. This would hinder the virus’s ability to reproduce; the virus would lose its

ability to enter the cell via endocytosis. This proposal was rejected due to the important functions

of nectin-1 and heparan sulfate. The receptors play essential roles in brain function, such as

synapse formation and cell adhesion12. Its modification could lead to unintended neurological

consequences. Furthermore, targeting the host receptors would not eliminate HSV-1 in the brain

but only prevents new infections. Our selected technology would be more effective because

instead of altering essential host receptors, it would target the virus directly and disable its ability

to reproduce.

A third potential approach would be to improve the immune response to HSV-1 in the

brain. The brain’s immune system could be boosted to better detect and suppress HSV-1

reactivation. This would entail enhancing activity in microglial cells, using immunotherapy to

neutralize HSV-1 in the brain, or changing cytokine signaling to reduce neuroinflammation.

However, there are several challenges with this approach. There exists a risk of excessive

inflammation through the overactivation of immune cells in the brain with neuroinflammation

being one of the catalysts of Alzheimer’s disease. Moreover, intensifying the immune response

might also lead to self-reactivity. Immune cells would mistakenly attack healthy cells such as

neurons and glial cells. This presents a serious health risk.

Discussion

Positives

1. Preventing HSV-1 triggered Alzheimer’s Disease

The immediate impact would be a huge breakthrough in the connection between HSV-1

and Alzheimer’s. This technology, if successful, could block the biochemical pathway that leads

to Alzheimer’s. For example, the pathway involving endoplasmic reticulum stress and the

unfolded protein response would cease to function, preventing the accumulation of amyloid-beta

and Alzheimer’s disease. Gene editing would provide a decreased risk to Alzheimer’s but would

only require a one-time treatment. Compared to antivirals that require lifelong usage, this

alternative is effective and convenient. If successfully implemented, CRISPR technology would

provide a preventative answer to Alzheimer’s disease for the millions of people that have herpes.

2. Advancing breakthroughs in gene-editing technology

This research could contribute to the growing field of CRISPR treatment, especially in

viral diseases. A successful gene editing of HSV-1 could establish a framework for using

CRISPR-Cas9 to approach other viral diseases. Furthermore, it could lead to developments in

overcoming the blood-brain barrier. The blood-brain barrier is a massive challenge when it

comes to treating neurological diseases as it prevents the delivery of therapeutic agents to the

brain. If there is success in penetrating the blood-brain barrier with the CRISPR components, it

could mean possible adaptations for other neurological conditions (e.g. Parkinson’s Disease).

3. Reducing healthcare costs

While CRISPR treatment for disease is currently extremely expensive, there is a

possibility that in the future CRISPR is integrated into the medical system as common practice.

This would allow for CRISPR treatment for the 3.7 billion people infected with HSV-1. The

implementation of CRISPR treatment to patients with herpes would then reduce the number of

Alzheimer’s patients and lessen the cost of Alzheimer’s care. In 2023, the annual cost of

Alzheimer’s care in the U.S. alone reached $321 billion.

Negatives

1. Moral and safety concerns in gene editing

CRISPR changes the genetic sequence of its targets. If CRISPR-Cas9 unintentionally

edits human genes involved in cognitive function, it would spell serious consequences such as

memory loss, personality changes, or other neurological dysfunctions. This raises ethical

concerns about the integration of CRISPR technology into our current medical fields.

2. Risk of HSV-1 developing resistance to CRISPR systems

While antibiotics have been proven to be a potent weapon against bacteria, there is a

possibility for HSV-1 to develop resistance mechanisms through mutations. HSV-1, like other

viruses, mutates over time. Theoretically, it could develop resistance that causes further CRISPR

targeting to be completely ineffective. As a result, it may create a need for re-engineering of

CRISPR strategies.

References

1. Alzheimer’s Disease International. (2021). World Alzheimer Report 2021. Retrieved from

https://www.alz.co.uk/research/WorldAlzheimerReport2021.pdf

2. Itzhaki, R. F., Lin, W.-R., Shang, D., Wilcock, G., Faragher, B., & Jamieson, G. (2003).

Herpes simplex virus in Alzheimer’s disease: the enemy within? Journal of Alzheimer’s

Disease, 5(6), 377–384. Retrieved from https://pubmed.ncbi.nlm.nih.gov/12915538/

3. Vassar, R. (2014). BACE1 inhibitor clinical trials: the rise and fall of the promise of

anti-amyloid therapies. Nature Reviews Drug Discovery, 13(2), 108–124. Retrieved from

https://doi.org/10.1038/nrd4256

4. Whitley, R. J., Kimberlin, D. W., & Roizman, B. (1998). Herpes simplex viruses. In D.

M. Knipe & P. M. Howley (Eds.), Fields Virology (3rd ed., pp. 2297–2360). Lippincott

Williams & Wilkins.

5. Cheng, Y., et al. (2022). Blood-brain barrier–penetrating single CRISPR-Cas9

nanocapsules for effective and safe glioblastoma gene therapy. Science Advances, 8(2),

eabo2358. Retrieved from https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9020780/

6. Takai, Y. (2004). Nectins and nectin-like molecules in cell adhesion, migration, and

polarization. Current Opinion in Cell Biology, 16(5), 572–579. Retrieved from

https://doi.org/10.1016/j.ceb.2004.08.008;

Sarrazin, S., Lamanna, W. C., & Esko, J. D. (2011). Heparan sulfate proteoglycans.

Annual Review of Biochemistry, 80, 929–955. Retrieved from

https://doi.org/10.1146/annurev-biochem-060909-152254

7. Jinek, M., Chylinski, K., Fonfara, I., Hauer, M., Doudna, J. A., & Charpentier, E. (2012).

A programmable dual-RNA–guided DNA endonuclease in adaptive bacterial immunity.

Science, 337(6096), 816–821. Retrieved from https://doi.org/10.1126/science.1225829

8. Daneman, R., & Prat, A. (2015). The blood–brain barrier. Cold Spring Harbor

Perspectives in Biology, 7(1), a020412. Retrieved from

https://doi.org/10.1101/cshperspect.a020412

9. Cheng, Y., et al. (2022). Blood-brain barrier–penetrating single CRISPR-Cas9

nanocapsules for effective and safe glioblastoma gene therapy. Science Advances, 8(2),

eabo2358. (See footnote 5 for URL)

10. Smith, G. P., & Petrenko, V. A. (1997). Phage Display. Chemical Reviews, 97(2),

391–410. Retrieved from https://doi.org/10.1021/cr960065d

11. Itzhaki, R. F., et al. (2003). Herpes simplex virus in Alzheimer’s disease: the enemy

within? Journal of Alzheimer’s Disease, 5(6), 377–384. Retrieved from

https://pubmed.ncbi.nlm.nih.gov/12915538/

12. Takai, Y. (2004). Nectins and nectin-like molecules in cell adhesion, migration, and

polarization. Current Opinion in Cell Biology, 16(5), 572–579. Retrieved from

https://doi.org/10.1016/j.ceb.2004.08.008;

13. Sarrazin, S., Lamanna, W. C., & Esko, J. D. (2011). Heparan sulfate proteoglycans.

Annual Review of Biochemistry, 80, 929–955. Retrieved from

https://doi.org/10.1146/annurev-biochem-060909-152254

14. Alzheimer's Association. (2023). 2019 Alzheimer’s Disease Facts and Figures. Retrieved

from https://www.alz.org/media/Documents/alzheimers-facts-and-figures-2019-r.pdf