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Introduction
Introduction
When someone says, “it’s the journey, not the destination that is important,” they usually are not talking about how medicines work. People often think that the most important part of medicines are the drugs themselves, but the way the therapies are delivered can be even more vital to treating a disease.
This study from Tel Aviv University, 'CRISPR-Cas9 genome editing using targeted lipid nanoparticles for cancer therapy,' explores the use of lipid nanoparticles (LNPs) as a delivery system for CRISPR/Cas9 gene editing with the intention of using it for cancer therapies. Recently, scientists have become increasingly interested in developing a variety of LNP systems for drug delivery in a variety of cancers as well as diseases like tuberculosis. Many of these studies have proven successful in being able to create systems that have increased bioavailability, longer retention levels, and lower hepatotoxicity and neurotoxicity levels than traditional chemotherapy strategies. This could improve the quality of life of patients undergoing cancer treatment as doses are less toxic and work for longer periods of time. Additionally, some of these studies have shown increased shelf-lives for their therapeutics. Overall, this study gives some promising insight into a future where cancer treatments are more accessible and effective.
Gene Editing Background
Gene Editing Background
The CRISPR/Cas9 system is a very valuable tool for altering the products of a cell at a molecular level. CRISPR, or clustered regulatory interspaced short palindromic repeats, was initially derived from bacteriophages and contains the codes needed to produce non-messenger RNAs. It is paired with a CRISPR associated nuclease (Cas), which essentially functions as a pair of scissors for DNA. There are a few different Cas variations but Cas9 is the most widely used.
Once inside the cell, the CRISPR/Cas9 pair is typically directed to the target location in the genome by a guide RNA (sgRNA) which allows scientists to edit any sequence near a PAM site. PAMs (protospacer adjacent motifs) are repetitive DNA sequences up to 5 base pairs that allow the sgRNA to bind and the gene editing to begin. Once bound to the target DNA, the Cas9 can create a double-stranded break, making the target useless.
Cells can repair DNA through the process of non-homologous end joining or homology directed repair. If scientists just want to stop gene function, they can rely on non-homologous end joining which attempts to fill in the breakage but has a very high error rate and is likely to create mutations. Conversely, if scientists want to insert DNA into a genome, they can pair the CRISPR/Cas9 system with a DNA template that will use homology directed repair to add in that additional DNA while the break is mended.
LNP Background
LNP Background
The different experiments done in the study support the overall claim by mixing Cas9 mRNA with sgRNA (single guide RNA) to make CRISPR LNPs (lipid nanoparticles) that usually has the cationic lipids, cholesterol and ssODN. In this case, there is lipid 1,6,8 and 10 in the LNP. Scientists looked at two aggressive cancer cell lines, one being murine GBM stem cells like 005 line (mice) and OV8, a type of cancerous ovarian cancer that is deadly to humans. The kinase that was looked at for both cancer cell lines is PLK1 which is vital for G2 and M phase in the cell cycle. When both were in vitro, PLK1 was induced so the cell cycle was stopped and the result was cell death.
There was an even more promising result when GBM was injected with mCherry and luciferase into the mouse hippocampus. When injected, the growth of the tumor was reduced significantly in the hippocampus of the mouse and is comparable to when PLK1 was injected into the mouse hippocampus. The study found that therapeutic strategies for a majority (metastatic) of the tumors are systematic not done by local administration. It is easiest to target and reduce the effect of the tumor by using systemically injected LNPs. These are coated with cell targeting antibodies which bind to a lipid anchored single chain antibody. Traditionally, LNPs are too big or get stuck in the liver/main organs and not taken by tumor cells.
Results
Results
This figure explains how this study is conducted. They looked at healthy patients and cancer patients. They took T cells from each and inserted the CRISPR nanoparticles in it to see the changes. The effects in both cases varied as in the case of cancer patients the inhibitory receptors were knocked out and the gene was editing successfully. In the case of healthy patients, they knocked out or removed genes related to HLA (important to the immune system) which helps defend cells against infection.
This study also showed us how CRISPR can be used to reduce tumors and increase the health of people who do have different types of cancer. There are some concerns about how to physically access the tumor site as well as who can receive the treatment. As for now, the results do work on mice and ovarian cancer which means that there is a good chance it will work on other patients and cancers too. This method of using CRISPR dramatically decreased the size of the tumor in the mouse model. We can use this result to possibly replicate this in humans. It can also boost the immune system and change how the body reacts to cancer.
Conclusion
Conclusion
This experiment aimed to investigate whether cLNPs (cationic lipid nanoparticles) can effectively deliver gene-editing components to disrupt a specific gene, PLK1, in two cancer cell lines: GBM 005 (a glioblastoma model) and OV8 (an ovarian cancer model). Both cancers are known for being highly aggressive and difficult to treat. GBM 005 comes from mice with gliomas, which are similar to deadly human brain cancers (GBM), characterized by being invasive and infiltrated by immune cells. OV8 is a model for high-grade ovarian cancer, resistant to chemotherapy and capable of spreading to form metastases.
The researchers used cLNPs as vectors to deliver a gene-editing tool called sgPLK1 into cancer cells. sgPLK1 works by destroying the PLK1 gene, which is essential for cancer cells to grow and divide.
These findings are useful to scientists and society at large as it provides a new way to reach disseminated tumors using LNPs, can be done with non vital kinases and is easy to deliver into a living organism. Cancer is a prevalent disease so improving how LNPs are delivered can help patients recover faster and shrink down the tumor significantly. The only concern is that repeated uses of this delivery system can lead to weakened immune responses and treatment failure so the Cas9 exposure should be limited and should also be delivered to the tumor site a limited amount of time.
Terms and Acronyms
Terms and Acronyms
LNP: Lipid Nanoparticle
CRISPR: Clustered Regulatory Interspaced Short Palindromic Repeats
Cas: CRISPR Associasted Nuclease
sgRNA: Single Guide RNA
PAM: Protospacer Adjacent Motif
PLK1: Polo-Like Kinase 1
OV8: Human ovarian cancer cell line
GBM: Glioblastoma Multiforme
HLA: Human Leukocyte Antigen
References
References
Duncavage, Eric J, et al. “Genomic Profiling for Clinical Decision Making in Myeloid Neoplasms and Acute Leukemia | Blood | American Society of Hematology.” Ash Publications, 24 Nov. 2022, ashpublications.org/blood/article/140/21/2228/486675/Genomic-profiling-for-clinical-decision-making-in.
Wang, S. W., Gao, C., Zheng, Y. M., Yi, L., Lu, J. C., Huang, X. Y., Cai, J. B., Zhang, P. F., Cui, Y. H., & Ke, A. W. (2022). Current applications and future perspective of CRISPR/Cas9 gene editing in cancer. Molecular cancer, 21(1), 57. https://doi.org/10.1186/s12943-022-01518-8
https://www.sciencedirect.com/science/article/abs/pii/S1044532317300398
https://www.sciencedirect.com/science/article/pii/S0749208119300129
https://onlinelibrary.wiley.com/doi/full/10.1002/anbr.202100109
https://www.nature.com/articles/s41565-020-0669-6
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8862238/
https://www.degruyter.com/document/doi/10.1515/dmpt-2018-0032/html
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