CRISPR-mediated APP C-terminal truncation may become a gene therapy for treating Alzheimer's disease by Atithi Sharma

DISCUSSION

atithisharma — 2022-11-29 02:40:19 UTC

CONCLUSION

atithisharma — 2022-11-29 02:40:22 UTC

The strategy of selective editing shows hopeful results as the authors were able to target and edit only the C-terminus of the APP. The CRISPR guided editing of the C-terminus resulted in the upregulation for the neuroprotective non-amyloidogenic pathway that might be used for the treatment of neurodegenerative disorders like Alzheimers.

HMB360 PRESENTATION

atithisharma — 2022-11-29 02:40:40 UTC

REFERENCES

atithisharma — 2022-11-29 02:40:46 UTC

Fellmann, C., Gowen, B. G., Lin, P.-C., Doudna, J. A., & Corn, J. E. (2017). Cornerstones of CRISPR-Cas in drug discovery and therapy. Nature Reviews. Drug Discovery, 16(2), 89–100. https://doi.org/10.1038/nrd.2016.238

Hornbeck, P., Winston, S. E., & Fuller, S. A. (2001). Enzyme-linked immunosorbent assays (ELISA). Current Protocols in Molecular Biology (Print), Chapter 11(1), Unit11.2–Unit11.2. https://doi.org/10.1002/0471142727.mb1102s15

Knopman, D. S., Amieva, H., Petersen, R. C., Chételat, G., Holtzman, D. M., Hyman, B. T., Nixon, R. A., & Jones, D. T. (2021). Alzheimer disease. Nature Reviews. Disease Primers, 7(1), 33–33. https://doi.org/10.1038/s41572-021-00269-y

NIH. (n.d.). App gene: Medlineplus genetics. MedlinePlus. Retrieved December 5, 2022, from https://medlineplus.gov/genetics/gene/app/#conditions

Struyfs, H., Van Broeck, B., Timmers, M., Fransen, E., Sleegers, K., Van Broeckhoven, C., De Deyn, P. P., Streffer, J. R., Mercken, M., & Engelborghs, S. (2015). Diagnostic Accuracy of Cerebrospinal Fluid Amyloid-beta Isoforms for Early and Differential Dementia Diagnosis. Journal of Alzheimer’s Disease, 45(3), 813–822. https://doi.org/10.3233/JAD-141986

Sun, J., Carlson-Stevermer, J., Das, U., Shen, M., Delenclos, M., Snead, A. M., Koo, S. Y., Wang, L., Qiao, D., Loi, J., Petersen, A. J., Stockton, M., Bhattacharyya, A., Jones, M. V., Zhao, X., McLean, P. J., Sproul, A. A., Saha, K., & Roy, S. (2019). CRISPR/Cas9 editing of App C-terminus attenuates β-cleavage and promotes α-cleavage. Nature Communications, 10(1). https://doi.org/10.1038/s41467-018-07971-8

Sun, X., Chen, W.-D., & Wang, Y.-D. (2015). Β-amyloid: The key peptide in the pathogenesis of alzheimer’s disease. Frontiers in Pharmacology, 6. https://doi.org/10.3389/fphar.2015.00221

Vu Nguyen, K. (2019). Β-amyloid precursor protein (APP) and the human diseases. AIMS Neuroscience, 6(4), 273–281. https://doi.org/10.3934/neuroscience.2019.4.273

FUTURE OUTLOOK

atithisharma — 2022-11-29 02:41:46 UTC

The off target effects analyzed in this experiment could not detect the small Double Stranded DNA breaks. So to further explore that there were no off target effects, more intense analysis that looks at the double stranded breaks thoroughly has to be performed.

The significance of editing the C-terminus of Amyloid Precursor Protein in vivo on Alzheimer’s disease needs to be further explored.

Sun J. has patented their Gene Editing-Based Method of Attenuating the Beta-Amyloid Pathway described in this article.

Roy, S., & Sun, J. (2022). Gene Editing-Based Method of Attenuating the Beta-Amyloid Pathway.

Sun J. continues researching in the underlying mechanisms and potential treatments for AD.

Shen, S., Liao, Q., Wong, Y. K., Chen, X., Yang, C., Xu, C., Sun, J., & Wang, J. (2022). The role of melatonin in the treatment of type 2 diabetes mellitus and Alzheimer’s disease. International Journal of Biological Sciences, 18(3), 983–994. https://doi.org/10.7150/IJBS.66871
Zhang, W., Xu, C., Sun, J., Shen, H.-M., Wang, J., & Yang, C. (2022). Impairment of the autophagy–lysosomal pathway in Alzheimer’s diseases: Pathogenic mechanisms and therapeutic potential. Acta Pharmaceutica Sinica. B, 12(3), 1019–1040. https://doi.org/10.1016/j.apsb.2022.01.008

ACKNOWLEDGEMENT

atithisharma — 2022-11-29 06:56:12 UTC

Atithi Sharma: Results, Discussion, Conclusion, Future Outlook
Haoyu Yang: Introduction, Methods, Future Outlook

INTRODUCTION: CRISPR/Cas9 guided gene editing

yhaoy2001 — 2022-12-06 21:25:02 UTC

Current practical limitations of CRISPR technology:

Canonical deletion of an entire gene by NHEJ is very likely to cause deleterious effects (Sun et al., 2019).
The procedure of point-mutation correction needs to be designed in mutation-specific basis (Sun et al., 2019).
Point-mutation correction only be applied to neurodegenerative diseases that are caused by inherited point mutations, which only covers <10% of all cases. (Sun et al., 2019)

Goal

yhaoy2001 — 2022-12-06 22:11:53 UTC

Apply CRISPR technology in treating neurodegenerative disease. More specifically, attenuate β-cleavage of APP and Aβ production by CRISPR-directed truncation of APP C-terminus.
Avoid deleterious effects. More specifically, preserve the normal physiological functions of APP.

Molecular hallmark of AD: Amyloid-β

yhaoy2001 — 2022-12-06 22:15:37 UTC

The aggregation of Amyloid-β (Aβ) plaques is a major neuropathological characteristic of AD (Knopman et al., 2021).

The neuroprotective α-cleavage and pathogenic β-cleavage of APP competitively inhibits each other. β-cleavage of APP predominates when APP is located in the plasma membrane, whereas α-cleavage predominates when APP is internalized into endosomes (Sun et al., 2019).

Experiment 1: Confirmation of CRISPR/Cas9 editing at APP C-terminus (Fig.1)

yhaoy2001 — 2022-12-06 22:17:38 UTC

Methods:

sgRNA selection based on Aβ-attenuating ability: APP-659 sgRNA.
Visualizing APP truncation by immunostaining:
- 22C11: APP N-terminus antibody
- Y188: APP C-terminus antibody
Analyzing effect of APP C-terminal truncation in β-cleavage pathway by western blot:
- CTF (C-terminal fragment): product of APP β-cleavage when γ-secretase is inhibited by GSI (γ-secretase inhibitor)

Experiment 2: Examination of effect of APP truncation in β/α pathways (Fig.2)

yhaoy2001 — 2022-12-06 22:33:08 UTC

Experimental groups: human iPSC-derived cell lines

WT/WT: wild type (control)
WT/APP^London: AD phenotype

Methods:

Immunostaining of APP C/N-terminus
- 2E9: APP N-terminus antibody
- Y188: APP C-terminus antibody
Western blot of sAPPα (product of α-cleavage)
ELISA (enzyme-linked immunosorbent assay) of β-pathway:
- Concentration of target molecule (antigen) measured by colour-change/fluorescence level.

Experiment 3: Analysis of off-target & deleterious effects (Fig.3)

yhaoy2001 — 2022-12-06 23:16:27 UTC

Methods:

Off-target analysis by T7 endonuclease assay
- Result: no CRISPR-editing in top 5 predicted off-target sites
Analysis in normal physiological roles of APP in cultured mouse hippocampus neurons:
- neurite outgrowth, axon-length, synaptic organization, or neuronal activity

Experiment 4: Investigation of APP β/α manipulation mechanisms in vivo (Fig.4, 5)

yhaoy2001 — 2022-12-06 23:28:20 UTC

Experimental Groups:

APP-659: CRISPR-mimicking (truncated C-terminus) construct
APP-T668A: mutation in APP C-terminal T668 residue
- Normal function of T668: co-localization with BACE1
APP-YENPTY: mutation in APP C-terminal YENPTY motif
- Normal function of YENPTY: internalization of APP into endosomes

Methods:

AAV9 carrying sgRNA delivered to neonatal mouse hippocampus / ventricles
Y188 immunostaining: confirmation of CRISPR/Cas9 editing (Fig.4)
BifC (Bi-molecular Fluorescence Complementation) assay: visualization of APP-BACE1 interaction (Fig.5a-d)
Internalization assay (APP N-terminus immunostaining) (Fig.5e)

Alzheimer's Disease (AD)

yhaoy2001 — 2022-12-06 23:54:57 UTC

https://alzheimer.ca/en/about-dementia/what-alzheimers-disease
The Alzheimer's Disease (AD) is a neurodegenerative disease commonly acquired in mid-life or late-life. The common symptoms of AD include impairment in memory, expressive speech, visuospatial processing and executive functions. (Knopman et al., 2021)

Current treatments for AD

yhaoy2001 — 2022-12-07 00:05:40 UTC

https://alzheimer.ca/en/about-dementia/how-can-i-treat-dementia/medications-approved-treat-alzheimers-disease

yhaoy2001 — 2022-12-07 00:15:08 UTC

Cholinesterase inhibitors:
- AD patients have lower level of acetylcholine. Cholinesterase inhibitors can prevent breakdown of acetylcholine.
NMDA receptor antagonists:
- AD patients have excess glutamate signalling. NMDA receptor antagonists can decrease glutamate signalling by blocking their receptors.

Both approaches can only temporarily ease AD symptoms, but are not cures for AD.

Experiment 1: .Using CRISPR/Cas9 editing to the APP C-terminus and manipulate the amyloid pathway in mouse cells

atithisharma — 2022-12-07 01:43:54 UTC

Main finding: mo-APP-sgRNA has selectively edited the C-terminus of the APP without affecting the N-terminus.

Significantly less Y188 C-terminus antibody signals in the neuro2a cells that are transfected with mouse-APP-sgRNA compared to cas9 only.
Significantly less CTFs signal in the mo-APP-sgRNA compared to the control-sgRNA but the signals in the N-terminus remained the same.

RESULTS

atithisharma — 2022-12-07 01:46:13 UTC

Experiment 2: The editing of APP C-terminus and the effects on the β/α pathway in human cells

atithisharma — 2022-12-07 02:15:19 UTC

Main Finding: The editing of the APP has reciprocal effects on β/α cleavage as the editing results in upregulation of α cleavage and downregulation of β cleavage

Human iPSC derived neurons were immunostained with the Y188 and tubulin antibodies, the APP expression is higher in the Control-sgRNA compared to hu-APP-sgRNA. This indicates that hu-APP-sgRNA has edited the APP C-terminus in human iPSC derived neurons.
In both the WT and London mutation group, there is an upregulation of sAPPα for the hu-APP-sgRNA transfected cells compared to control-sgRNA which indicates the upregulation of non-amyloidogenic pathway.
In both the WT and London mutation group, when ELISA and western blot was performed, there is a downregulation of sAPPβand Aβ 40/42 for the hu-APP-sgRNA transfected cells compared to control-sgRNA. This indicated the downregulation of the amyloidogenic pathway.

Experiment 3: Neurophysiology effects of editing the APP and the analysis of off targets

atithisharma — 2022-12-07 02:55:10 UTC

Main Finding: No significant effect was detected for the editing of APP C-terminus on the N-terminus neuronal physiology.

In the cultured neurons, both cas9 only and mo-APP-sgRNA had similar results for the N-terminal but the neurons transfected with AAV9-Cas9 had higher Y188 antibody in the CTFs compared to the neurons transfected with AAV9-sgRNA.
The normalized axon length has no significant difference for both the Cas9 only and the mo-APP-sgRNA.
The cas9 only and mo-APP-sgRNA transfected neurons were stained with the presynaptic marker VAMP2. The presynaptic density was compared for the mo-APP-sgRNA and Cas9 only transfected neurons, the result had no significant difference. This indicates that there is no significant neuronal physiological difference between the edited and unedited APP C-terminus.

Experiment 4: In vivo editing of the APP C-terminus

atithisharma — 2022-12-07 03:08:28 UTC

Main Finding: The mo-APP-sgRNA has successfully edited the C-terminus in vivo (mouse)

Immunostaining with the APP Y188 antibody was performed, and there wasless fluorescence in the APP-sgRNA transfected mouse compared to Cas9 only and uninfected mouse. This shows the evidence of APP editing in vivo in mouse.

Experiment 4: The mechanism of CRISPR guided editing of the APP C-terminus and manipulation of α/β cleavage

atithisharma — 2022-12-07 03:15:39 UTC

Main Finding: There was less interaction between APP and BACE-1 for the AAV9-sgRNA transfected neurons.

In the interaction of APP/BACE-1 between the wildtype and the AAV9-sgRNA transfected neurons, the fluorescence is detected much more in the wildtype compared to the transfected neurons which indicates that there was less interaction between APP and BACE-1.
The internalization assay showed that APP are mostly internalized into endosomes in WT neurons; where as APP in APP659 (crispr-mimicking) neurons mostly remains in plasma membrane, suggesting that C-terminal truncation likely attenuates β-cleavage by preventing APP internalization.

Findings

atithisharma — 2022-12-07 04:05:30 UTC

Using Crispr/Cas9 techniques to selectively edit the amyloid precursor protein (APP) at the extreme C-terminus, the authors were able to manipulate the amyloid pathway by decreasing the APP-β-cleavage and Aβ production, while up-regulating APP-α-cleavage. This is a significant finding as APP is linked to many different neurodegenerative diseases such as Autism, fragile X syndrome and many more. The selective editing techniques can be used in creating a gene therapy for neurodegenerative diseases. (Vu Nguyen, K., 2019)

Challenges

atithisharma — 2022-12-07 04:09:38 UTC

Maintaining the normal functions of the endogenous proteins as it is responsible for the physiological roles.
Ensuring there are no off target effects
Maintaining the function of N-terminus as the goal of this experiment was to edit only the C-terminus of APP

Strength and Weakness

atithisharma — 2022-12-07 04:10:35 UTC

Strength:

Selective gene editing approach that edits the APP while maintaining its neurophysiological functions
Experiment in performed in both in cultured cells and in mouse
Experiment was performed in an “AD-like setting”
Multiple experiments performed to confirm that APP C-terminus was edited without off target effects

Weakness:

There is no data for the long term effects of this editing strategy so the preservation of the physiological function in the long term can not be concluded.
The off target effects analyzed in this experiment could not detect the small Double Stranded DNA breaks.
Although the experiment was performed in an “AD-like setting” and in-vitro, the significance of this experiment for in vivo Alzheimer's can not be determined until further experiments are performed.

METHODS

atithisharma — 2022-12-07 04:17:58 UTC

Information on APP normal function and Alzheimer's Disease

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