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Fighting Muscle Atrophy with Gene Editing

This approach is successful in mouse models.

Muscle DNAMuscle DNA
 

In a new study published in Journal of Nanobiotechnology, researchers prevented muscle atrophy both in vitro and in vivo by delivering a CRISPR-Cas9 gene editing system packaged in engineered extracellular vesicles [1].

When just using it doesn’t quite cut it

Aging is accompanied by the loss of muscle mass, also known as sarcopenia, which deteriorates up to 50% of muscle tissue by the age of 80 [2]. Age-related muscle atrophy is associated with a number of pathological conditions, including osteoporosis, and ultimately leads to frailty.

Bones adapt to muscle load: in other words, the bigger the muscles, the stronger the bones. Integrating strength training into a workout routine is currently the only effective way to offset sarcopenia. Therefore, other therapeutic approaches are being explored, including gene therapy that targets microRNAs.

MicroRNAs are non-coding RNAs regulating the expression of their target messenger RNAs. One such microRNA gaining attention in aging research is miR-29b, a regulator of extracellular matrix proteins that is also involved in the differentiation of osteoblasts, bone-building cells. This microRNA was shown to be upregulated in a variety of aging-associated processes [3]. Therefore, reducing miR-29b is being investigated as an anti-aging therapeutic target.

miR-29b inhibitors are already commercially available, although their use is limited due to their unpredictable side effects and toxicity. Previously, a CRISPR/Cas9 gene editing system targeting miR-29b packaged into lentivirus and adeno-associated viruses (AAV) was shown to suppress this microRNA and protect against muscle atrophy in a mouse model [4].

In this study, the researchers packaged miR-29b-targeting CRISPR-Cas9 into extracellular vesicles instead of viral particles. Extracellular vesicles are lipid-bilayered particles naturally secreted by cells to deliver various molecules to recipient cells. They are considered a superior option compared to viral delivery in terms of immune response and packaging capacity.

Constructing the delivery system

First, the researchers constructed the delivery system by packaging the Cas9 protein, which is the molecular ‘scissors’, along with a small guide RNA that recognizes miR-29b and guides Cas9 to target it, into engineered extracellular vesicles (EVs). In addition to their gene editing cargo, the EVs contained a glycoprotein derived from a virus to make them more efficient at entering the target tissue’s cells.

These EVs were produced by introducing several plasmids encoding the sequences required for the production of EVs into a cell culture, followed by several centrifugation steps to get rid of other cell components and isolate the EVs. Various techniques were then used to confirm that the EVs contained the expected cargo and demonstrated the characteristic morphology.

The researchers then checked if the engineered EVs could achieve their goal of reducing the expression of miR-29b. They treated muscle cells in culture with the EVs and confirmed that the EVs disrupt the expression of miR-29b through gene editing. Importantly, the silencing effect was achieved in a short period of time (24 hours) and in almost all of the cells.

The delivery system in action

Next, the researchers induced muscle atrophy in a cell culture by applying two different drugs, dexamethasone and angiotensin II, and the cytokine TNF-α. They confirmed that the application of the engineered EVs reversed muscle atrophy in all these in vitro models as evidenced by increased muscle fiber diameter and reduced expression of muscle atrophy markers.

Finally, the engineered EVs were injected intramuscularly into eight-week-old male mice with muscle atrophy. The researchers used two mouse models of muscle atrophy: immobilization-induced and denervation-induced. For the former, the hind limbs of mice were fixed with screws to achieve immobilization. For the latter, the mice had their sciatic nerves cut. This prevented these mice from sending nerve signals to their legs, leading to muscle atrophy.

The EV injections were then performed into the gastrocnemius leg muscle seven times, spread over a five-week period following either the immobilization or the sciatic nerve surgery.

In both models, the EVs were successfully delivered into the muscle tissue of the mice, inhibiting the expression of miR-29b without any side effects. Most importantly, the EVs increased the leg muscle weight and the muscle fiber size in these in vivo models of muscle atrophy. In addition, the expression of muscle atrophy markers was reduced while the expression of IGF1 and PI3K was restored in EV-treated mice.

Abstract

Muscle atrophy is a frequently observed complication, characterized by the loss of muscle mass and strength, which diminishes the quality of life and survival. No effective therapy except exercise is currently available. In our previous study, repressing miR-29b has been shown to reduce muscle atrophy. In our current study, we have constructed artificially engineered extracellular vesicles for the delivery of CRISPR/Cas9 to target miR-29b (EVs-Cas9-29b). EVs-Cas9-29b has shown a favorable functional effect with respect to miR-29b repression in a specific and rapid manner by gene editing. In in vitro conditions, EVs-Cas9-29b could protect against muscle atrophy induced by dexamethasone (Dex), angiotensin II (AngII), and tumor necrosis factor-alpha (TNF-α). And EVs-Cas9-29b introduced in vivo preserved muscle function in the well-established immobilization and denervation-induced muscle atrophy mice model. Our work demonstrates an engineered extracellular vesicles delivery of the miR-29b editing system, which could be potentially used for muscle atrophy therapy.

Conclusion

This study shows that targeting miR-29b using an EV-packaged gene editing system is an effective and safe approach to treat muscle atrophy in mouse models. Importantly, the EVs were derived from muscle cells, so they did not trigger an immune response when injected into skeletal muscles. The authors acknowledge that some molecules from the cells used to produce the EVs could have been packaged into the EVs; therefore, the assumption of safety might be false, which requires further investigation.

Currently, one of the biggest challenges of gene therapy is tissue-specific delivery. By engineering EVs to contain surface proteins that recognize specific cells, it may be possible to achieve targeted delivery. Hopefully, future studies will explore this venue of translational research.

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Literature

[1] Chen, R. et al. Delivery of engineered extracellular vesicles with miR-29b editing system for muscle atrophy therapy. J. Nanobiotechnology 20, 1–16 (2022).

[2] Walston, J. D. Sarcopenia in older adults. Curr. Opin. Rheumatol.24, 623–627 (2012).

[3] Zhao, W., Cheng, L., Quek, C., Bellingham, S. A. & Hill, A. F. Novel miR-29b target regulation patterns are revealed in two different cell lines. Sci. Rep. 9, 17449 (2019).

[4] Li, J. et al. CRISPR/Cas9-Mediated miR-29b Editing as a Treatment of Different Types of Muscle Atrophy in Mice. Mol. Ther. 28, 1359–1372 (2020).

About the author
Larisa Sheloukhova

Larisa Sheloukhova

Larisa is a recent graduate from Okinawa Institute of Science and Technology located in one of the blue zones. She is a neurobiologist by training, a health and longevity advocate, and a person with a rare disease. She believes that by studying hereditary diseases it’s possible to understand aging better and vice versa. In addition to writing for LEAF, she continues doing research in glial biology and runs an evidence-based blog about her disease. Larisa enjoys pole fitness, belly dancing, and Okinawan pristine beaches.
  1. 8080abc
    July 21, 2022

    Finally, we have noticed microRNA! How difficult it is to accept the fact that the body ages in two ways:

    1. using programs that are controlled by microRNA

    2. by the extracellular matrix (accumulation of crosslinks, reduction of elastin content, etc.), but these processes also seem to be regulated by microRNA (except for glucosepane crosslinks).

    Due to these factors, the number of stem cells decreases, the work of mitochondria deteriorates, telomeres sharply shorten, etc. It seems that all this is a consequence of the implementation of the aging program in the body, + modification of matrix proteins over time, which contribute to aging.

  2. Phil Goetz
    August 2, 2022

    We’ve had similarly effective gene-therapy treatments for MD for 30 years, but none of them have ever been used except in tiny clinical trials. This one will also go nowhere until the FDA is reformed to use a cost-benefit analysis for approval.

    • Phil Goetz
      August 2, 2022

      Or maybe only slightly over 20 years. I forget now when the first effective gene-therapy treatment for MD was tried. Sometime in the 1990s.

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