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Using CRISPR for a novel senolytic therapy

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Jamila Oct 06, 2020
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As mentioned in this session, senescent cells could become resistant to senolytic drugs at some point. So this sparked my imagination! I wondered if we could use CRISPR as a non-traditional senolytic therapy?

Basically, the idea is that CRISPR would be used to target specific gene(s) to drive apoptosis in senescent cells specifically. For example, the senolytic drug navitoclax inhibits BCL-XL, and this causes apoptosis in senescent cells. Using a similar approach, we could downregulate the BCL2-like 1 gene (encodes BCL-XL) with CRISPR repressors, and this would hopefully induce apoptosis in senescent cells.

CRISPR Technology
There are various CRISPR technologies out there. There is the traditional gene editing, base editing, transcriptional regulation, etc. CRISPR based transcriptional regulation doesn't permanently change the DNA; it regulates the gene expression levels of a specific gene. This is contrary to traditional CRISPR gene editing, which makes permanent changes in the genetic code. CRISPR is a versatile tool; it has many applications and can be used to target multiple genes at once.

CRISPR based transcriptional regulation uses a dead Cas (dCas) with a transcriptional activator or repressor attached to it. A specific guide RNA is used to guide the dCas to the specific gene. The specific gene will be upregulated or downregulated depending on the transcriptional factor attached to dCas.

The Possible Target Genes
We might be able to remove senescent cells by targeting SCAPs or SASPs related pathways:
  • BCL-2 family (BCL-2, BCL-XL, etc.)
  • p53/p21/serpines
  • PI3K/AKT
  • Others
Here are some things to think about:
  1. Which gene(s) should be targeted?
  2. How will the senescent cells be specifically targeted?
  3. Are CRISPR based transcriptional regulators the best option to go with?
  4. Can genes be upregulated and downregulated simultaneously with CRISPR?
  5. How long would the CRISPR components stay in the body?
  6. Suppose things went wrong like for example healthy cells being targeted. How could we ensure safety?
What do you think of the idea? Do you think this idea could be further upgraded?

[1]Yosef, Reut, et al. "Directed elimination of senescent cells by inhibition of BCL-W and BCL-XL." Nature communications 7.1 (2016): 1-11.

[2]Adli, Mazhar. "The CRISPR tool kit for genome editing and beyond." Nature communications 9.1 (2018): 1-13.

[3]Maeder, Morgan L., et al. "CRISPR RNA–guided activation of endogenous human genes." Nature methods 10.10 (2013): 977-979.

[4]Kirkland, James L., and Tamara Tchkonia. "Cellular senescence: a translational perspective." EBioMedicine 21 (2017): 21-28.

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General comments

Antonio Carusillo
Antonio Carusillo4 years ago
In principle, the idea is well thought out. However, although CRISPR/Cas system has revolutionized the world of genetic engineering ( just today 7.10.2020 Emmanuelle Charpentier and Jennifer A. Doudna had been awarded the Nobel Price in Chemistry for bringing CRISPR to mammalian cells ) it still has some limitations. In particular, if we envision to deliver CRISPR in vivo – meaning by injecting it directly into the patient – we will face two main challenges: - How many cells can we target? To have a proper effect we will need to target more than half of the senescent cells ( at least ) to see an effect like tissue rejuvenation - How can we make sure to target only the senescent cells and not healthy cells? So imagine you are injecting CRISPR into the body. How can you control were CRISPR will end up? How to make sure that CRISPR only acts on the senescent cells and not for example on healthy T cells. Currently, some labs are developing a way to deliver CRISPR in a specific cell type (https://pubs.acs.org/doi/abs/10.1021/jacs.9b11638) but they are far away from a clinical application anytime soon. Nevertheless, CRISPR may still be used to identify new analytics. I am glad you brought up the example of a drug. CRISPR is being used quite extensively from companies to screen for new target genes against which design a drug. ( in jargon they say “ druggable genes” ). So imagine having a 96-well plate ( meaning a plate where there are 96 spots hosting cells ) with inside your senescent cells. At this point, via high-throughput processes, you can deliver CRISPR to each one of the 96 spots and you can target 96 genes ( 1 per spot ). You can extend this experiment to 2 or 3 or 10 plates like this being able to screen for hundreds of genes pretty fast. This is the possible cause while the Cas9 ( the molecular scissor of CRISPR ) is always the same, for each well you change the guide RNA ( the molecular radar that guides CRISPR toward its target). How would you screen? For example, you may have in the same well healthy cells and senescent cells. You deliver CRISPR and you look in which well only the healthy cells survived and the senescent cells died out. You can then track back which gene was hit cause the guide RNA has a “barcode”. So let´s say that you find that in well number 34 all the healthy cells are alive but the senescent cells are dead, you can tell exactly which gene you targeted in well 34. Such an approach is known as CRISPR-library screening ( https://pubmed.ncbi.nlm.nih.gov/29158600/ ) From an experiment like this, you can then identify different genes. At this point, the company can synthesize compounds aiming to block this gene – like the one you mentioned – and the test is in an animal model and a later stage in human. Overall, CRISPR can indeed be a powerful tool to address ageing but in this case, it will be a mean, not the main actor. So CRISPR can be used to identify new targets and speed up the development of more efficient senolytics!
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Jamila 4 years ago
Thanks for the feedback. 😊 It was about time Doudna and Charpentier were awarded the Nobel prize for their outstanding contributions; it's incredible!! I definitely agree with you. CRISPR does have its limitations. It is far from perfect, but the possibilities with the technology are unlimited. The challenges you bring up are crucial for this idea to work. As you said, we need to find out how many senescent cells have to be removed for a therapeutic benefit. Perhaps, a small but targeted removal of senescent cells would still produce beneficial results. In that case, we might not need to target many senescent cells in the body. But yes, all these fantastic ideas definitely should be tested out. Perhaps, we could specifically target senescent cells using a receptor-mediated delivery method; this should be explored in the future. [1] The CRISPR-library screening approach you describe with the senescent and healthy cells is a fantastic idea! It would help to find new targets for senolytic drugs – because drug resistance is an issue. [2] It can also help researchers determine which genes to target with CRISPR in the future (providing my idea is viable). Many many years ago, I wouldn't have thought we could precisely target genes and DNA bases as we can with CRISPR nowadays. I believe the capabilities we see with CRISPR right now are just the tip of the iceberg. References 1.Kang, Chanhee. "Senolytics and senostatics: a two-pronged approach to target cellular senescence for delaying aging and age-related diseases." Molecules and Cells 42.12 (2019): 821. 2.Rouet, Romain, et al. "Receptor-mediated delivery of CRISPR-Cas9 endonuclease for cell-type-specific gene editing." Journal of the American Chemical Society 140.21 (2018): 6596-6603.
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Shubhankar Kulkarni
Shubhankar Kulkarni4 years ago
How about combining the two different approaches here? - Utilizing CAR T cells (Antonio's suggestion - https://brainstorming.com/sessions/how-can-we-overcome-the-problem-of-resistance-of-the-senescent-cells-to-senolytics/139) to target and identify the senescent cells and using CRISPR to downregulate the target genes (Jamila's idea)? Based on what I understand, Chimeric Antigen Receptor (CAR) T cells are re-engineered T cells to target specific cells. However, instead of mounting a full-blown immune response, they could induce apoptosis in the senescent cells using CRISPR. This will eliminate the target recognition problems associated with CRISPR and immune hyperactivity (toxicity towards healthy cells) observed with a full-blown immune response.
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Jamila 4 years ago
Good thinking, Shubhankar. Previously I've heard of researchers using T cells to "carry" drugs to cancer cells. Applying the same principles here is an excellent thought. [1] Instead, the T cells will be carrying CRISPR components, and then directed towards senescent cells. This could solve the challenge of targeting senescent cells only. We would need a highly specific target on senescent cells so that healthy cells aren't targeted. High specificity will help reduce the off-target effects on healthy cells and, subsequently, organ failure. [2] Reference: 1.Siriwon, Natnaree, et al. "CAR-T cells surface-engineered with drug-encapsulated nanoparticles can ameliorate intratumoral T-cell hypofunction." Cancer immunology research 6.7 (2018): 812-824. 2.Wäsch, Ralph, Markus Munder, and Reinhard Marks. "Teaming up for CAR-T cell therapy." haematologica 104.12 (2019): 2335.
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Antonio Carusillo
Antonio Carusillo4 years ago
The idea of delivery CRISPR nested in the CAR-T cells could be a nice concept. However, I wonder if this would be possible as it is not only the Cas9 protein to be delivered but also the sgRNA. Even if such an obstacle should not be a big deal, we should be thinking about the advantage of co-delivering CRISPR. For example, if CRISPR is meant to do the same job as the CART cells, then we do not really need CRISPR. For the reasons said above, CART cells will be more efficient at targeting the senescent cells and triggering their death rather than CRISPR. So is it worth it? On the other hand, CRISPR may be used as a tool to improve CART cells themselves like was done by Dr Carl June (https://science.sciencemag.org/content/367/6481/eaba7365) for example by rendering them less prone to exhaustion, a process because of which T cells stop dividing and killing. Another scenario can be that upon killing the senescent cells we observe some side effects perhaps as a consequence of the release from the cells of cytosolic signals, the so-called Senescent-Associated Secretory Phenotype (https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4166495/) which may affect the surrounding healthy cells. In this sense, we may devise a strategy where for example we can use CRISPR to transiently silence genes responsive to SASP in the healthy cells. So, personally, if we do not foresee any side effect coming from the killing of the senescent cells, I would still use CRISPR as a tool rather than as the main actor either to potentiate the CART cells or to ameliorate side effects. The more elements you add to the puzzle and the more complicated it gets. If you think about all the current clinical trials - involving gene and cell therapy - there are very few elements involved and the approach is kept as simple as possible. In an in vitro system it may work, but in complex human settings the fewer elements to check the better.
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