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Can exosomes be used to treat Sarcopenia?

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Subash Chapagain
Subash Chapagain Nov 21, 2020
What novel treatment strategies can we develop for treating age-related muscular dystrophy?

Considered one of the hallmarks of the ageing process, Sarcopenia is a geriatric condition that is characterized by a progressive loss of muscle mass, strength and function, affecting balance and eventually the overall ability to perform daily tasks of living. The European Working Group on Sarcopenia defines the disease as “progressive and generalized loss of skeletal muscle mass and strength with a risk of adverse outcomes such as physical disability, frailty, poor quality of life, and death .”

Recognized as a distinct disease condition late in 2016 only, different studies show that the average prevalence of sarcopenia in older adults aged 60-70 years is at 5-13%, amounting to 11-50% in people aged 80 and older . Age-related mitochondrial dysfunction and age-associated inflammation are deemed to be the core molecular causes of sarcopenia, though there are other subtle mechanisms involved in the manifestation of the condition .

Current treatment strategies for Sarcopenia include nutritional and exercise interventions, but both of these approaches have been controversial in regard to their clinical benefits. When it comes to treating older people, exercise (that heavily includes resistance training and physical activities ) naturally becomes a less attractive option. Potential drugs for sarcopenia include testosterone and anabolic steroids, myostatin antibodies, activin receptor antibodies and anamorelin; however fully unrealized. The limitations of exercise and pharmacological interventions have made it imperative to search for novel approaches to dealing with the age-related muscular dystrophy accompanying the condition of Sarcopenia.


The exosomal connection

Through some exciting research, exosomes have come up as one of the possible tools for treating sarcopenia. Exosomes are a homogeneous population of endogenous vesicles (lipid compartments) with a diameter of 30-200nm, derived from inward budding of the multivesicular body membrane and secreted into the outside of the cells upon fusion of multivesicular body and plasma membrane. When the study investigated how exercise helped attenuate sarcopenia, it was found that heat-shock protein 60 (Hsp)-60 bearing exosomes were released after physical training, that actively increased PGC1α (peroxisome proliferator‐activated receptor-gamma coactivator 1 alpha) .

Since exosomes have already been deemed through research possible intervention strategies for other diseases like type 2 diabetes and age-related bone loss and osteoarthritis , it is plausible that in the context of sarcopenia, specific circulating miRNA or other modulators included in exosomes may be useful in tackling the otherwise untreated condition. For instance, EV (extracellular vesicles)-miR-133b and EVs-miR-181-1-5p were also observed to be upregulated post-exercise, possibly related to muscle remodelling and homeostasis . More recently, it has also been found that muscle-derived EVs that are loaded with senescence-associated miRNAs, such as miR-34a were able to induce cellular senescence in bone stem cells, highlighting their connection in the ageing process. Since exosomes belong to EVs and they also carry significant information (and molecules) as DNAs, miRNAs and circular RNAs, one possible approach to using them for sarcopenia treatment would be to engineer them to deliver specific signal molecules including myogenic growth factors (for example IGFs, FGF-2 and hepatocyte growth factor).

On the grounds of this aforementioned relevant evidence and examples, could we effectively use exosomes and exosomal engineering to at least reduce the burden of sarcopenia if not totally cure it? What other novel strategies can we think of in terms of research prospects for fighting age-related muscular dystrophy?

[1]Cruz-Jentoft AJ, Landi F, Schneider SM, Zuniga C, Arai H, Boirie Y, Chen LK, Fielding RA, Martin FC, Michel JP, Sieber C, Stout JR, Studenski SA, Vellas B, Woo J, Zamboni M, Cederholm T: Prevalence of and interventions for sarcopenia in ageing adults: a systematic review. Report of the International Sarcopenia Initiative (EWGSOP and IWGS). Age Ageing 2014;43:748-759.

[2]von Haehling S, Morley JE, Anker SD. An overview of sarcopenia: facts and numbers on prevalence and clinical impact. J Cachexia Sarcopenia Muscle 2010;1:129–133.

[3]Lo, J. H.-, U, K. P., Yiu, T., Ong, M. T.-, & Lee, W. Y.-. (2020). Sarcopenia: Current treatments and new regenerative therapeutic approaches. Journal of Orthopaedic Translation, 23, 38–52. https://doi.org/10.1016/j.jot.2020.04.002

[4]Naranjo J, D, Dziki J, L, Badylak S, F: Regenerative Medicine Approaches for Age-Related Muscle Loss and Sarcopenia: A Mini-Review. Gerontology 2017;63:580-589. doi: 10.1159/000479278

[5]Barone R, Macaluso F, Sangiorgi C, Campanella C, Marino Gammazza A, Moresi V, et al. Skeletal muscle heat shock protein 60 increases after endurance training and induces peroxisome proliferator‐activated receptor gamma coactivator 1 alpha1 expression. Sci Rep 2016;6:19781.

[6]Safdar A, Saleem A, Tarnopolsky MA. The potential of endurance exercise‐derived exosomes to treat metabolic diseases. Nat Rev Endocrinol 2016;12:504–517.

[7]Murphy C, Withrow J, Hunter M, Liu Y, Tang YL, Fulzele S, et al. Emerging role of extracellular vesicles in musculoskeletal diseases. Mol Aspects Med 2018;60:123–128.

[8]Guescini M, Canonico B, Lucertini F, Maggio S, Annibalini G, Barbieri E, et al. Muscle releases alpha‐sarcoglycan positive extracellular vesicles carrying miRNAs in the bloodstream. PLoS ONE 2015;10:e0125094

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Creative contributions

Feedback on the Proposed Usage of Exosomes

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Andrew Pan Nov 24, 2020
While exosomes are and remain an exciting field of research, especially in the space of drug delivery, the heterogeneity of exosomes and their contents are large barriers towards clinical translation. Codiak biosciences is a prominent player in this field. What may be useful is a bottom-up approach of manufacturing lipid nanoparticles with beneficial properties from exosomes (whether they be specific lipid composition or a peptide on the exosome surface which enhances cell/tissue specific targeting).

Using CRISPR to target Myostatin Gene

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Antonio Carusillo
Antonio Carusillo Nov 24, 2020
Sarcopenia defines a condition of progressing muscle weakening and wasting triggered by ageing and sometimes associated with certain cancer types and diabetes.

Due to my particular interest in genetic engineering, I will propose as a first possibility the use of CRISPR/Cas9 technology.
We have already discussed CRISPR as tool to address cell senescence in previous sessions so I will just highlight again that this tool allows for the targeted introduction of DNA Double-Stranded Break within a genetic sequence of interest.

Hereby, CRISPR/Cas9 can be considered as a molecular scissor that we can program to cut – virtually – any desired sequence.

Before we move to Sarcopenia, it is important to highlight that CRISPR based strategies are being already explored in the treatment of conditions affecting Skeletal Muscles. In particular, CRISPR is being tested for the treatment of Duchenne Muscle Dystrophy (DMD) . Although DMD is not the focus of this session there is some important information that we can derive from it:
  • CRISPR is already being tested in muscle cells
  • There are already in place plenty of protocols to deliver CRISPR in vivo to skeletal muscle cells
This means that – theoretically – the only thing that we need to do is to reprogram CRISPR to target a specific gene instead of the dystrophin ( targeted in the DMD treatment).

So what gene should we target in such contest ?

An attractive one would be the MSTN gene which encodes for the Myostatin protein which:

  1. has been involved in the activation of the SMAD/SMAD3 signalling which is involved in priming muscle atrophy
  2. Also, MSTN deficient young patients are characterized by muscle hypertrophy .
These observations make MSTN a suitable candidate.

Such concept has been already explored in vivo by two different research groups which used almost the same approach: they delivered the CRISPR with a single non integrating viral vector specific for skeletal muscle cells .
This is possible to achieve cause the Adeno Associated Virus 8 (AAV8) is known to have a specific tropism for muscle cells and it has been already tested in the contest of DMD treatment.

So, what the researched did was to cut the MSTN gene aiming to abrogate the Myostatin expression.

In the first paper, in particular, they could reach a fairly high gene disruption roughly between 25% and 30%. After that, they had a closer look at the left thigh muscle injected with CRISPR and compared it to the control mice.
Amazingly they could observe an increase in the size of the quadriceps and adductor, indicating an increase in muscle growth. Such shreds of evidence were confirmed by analyzing the muscle fibres where they could detect an increase in their numbers and size. This was mainly due to the reduction of the Myostatin-axis which in consequence resulted in lower expression of the proteasome associated ubiquitin-protein ligase involved in the degradation of myogenic factors like MyoD, MyoG and Pax7 .
The second paper ,although with a lower editing efficiency, could also show MSTN editing as a promising approach, in particular, together with a reduced fibre muscle waste, they observed that edited mice improved their forearm grip strength by 25% compared to the control mice.

Overall, both papers seem to suggest that SMNT knock-out could be a possible therapeutic strategy to address sarcopenia.

A possible drawback of such a strategy could be the possibility to introduce modifications in undesired genetic loci ( a.k.a off-targets) which may result in hard to predict side effect as well as gross chromosomic rearrangements.

For such reasons, we may need to:
  1. check for possible off-targets as well as DSB induced gross rearrangements
  2. if off-targets are found, we may have to devise strategies to reduce off-targets. Some of those have been described in this session
  3. use DSB-free alternatives like base-editing or prime editing. These have been described also in the session linked above. However, at the moment the size of base-editors and prime-editors are prohibitive to be packaged in a single viral vector. To this regard, technological advancements will be required for the delivery of these tools in vivo as well.

[1]Enhanced CRISPR-Cas9 correction of Duchenne muscular dystrophy in mice by a self-complementary AAV delivery system BY YU ZHANG, HUI LI, YI-LI MIN, EFRAIN SANCHEZ-ORTIZ, JIAN HUANG, ALEX A. MIREAULT, JOHN M. SHELTON, JIWOONG KIM, PRADEEP P. A. MAMMEN, RHONDA BASSEL-DUBY, ERIC N. OLSON SCIENCE ADVANCES19 FEB 2020 : EAAY6812

[2]Cohen, S, Nathan, JA and Goldberg, AL (2015). Muscle wasting in disease: molecular mechanisms and promis- ing therapies. Nat Rev Drug Discov 14: 58–74.

[3]Lee, YS, Huynh, TV and Lee, SJ (2016). Paracrine and endocrine modes of myostatin actin. J Appl Physiol (1985) 120: 592–598.

[4]Prevention of Muscle Wasting by CRISPR/Cas9-mediated Disruption of Myostatin In Vivo Wei, Yuda et al. Molecular Therapy, Volume 24, Issue 11, 1889 - 1891

[5]Weng, S., Gao, F., Wang, J. et al. Improvement of muscular atrophy by AAV–SaCas9-mediated myostatin gene editing in aged mice. Cancer Gene Ther (2020). https://doi.org/10.1038/s41417-020-0178-7

[6]Systemic gene transfer reveals distinctive muscle transduction profile of tyrosine mutant AAV-1, -6, and -9 in neonatal dogs Hakim, Chady H et al. Molecular Therapy - Methods & Clinical Development, Volume 1, 14002

[7]Langley B, Thomas M, Bishop A, Sharma M, Gilmour S, Kambadur R. Myostatin inhibits myoblast differentiation by down-regulating MyoD expression. J Biol Chem. 2002 Dec 20;277(51):49831-40. doi: 10.1074/jbc.M204291200. Epub 2002 Sep 18. PMID: 12244043.

Using Biological Scaffolds

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Subash Chapagain
Subash Chapagain Nov 26, 2020
The skeletal muscle tissue has the capacity to regenerate after an acute injury, a property of these tissues by the virtue of the activity of satellite cells (introduced in the above contributions). Nevertheless, the microenvironmental niche conditions play a determining role in the proliferative capacity of the satellite cells and hence influences the regeneration of skeletal muscle as the whole, fully functioning tissue.

If the inherent signals that are relayed to and from the injured/damaged cells to the healthy cells are lost, then the regenerative capacity is disrupted. It has been evident that changing the environment of aged myogenic progenitor cells can enhance skeletal muscle regeneration. The natural microenvironment of healthy extracellular matrix in the form of biologic scaffolds has been clinically reported to promote myogenesis in patients that suffered the volumetric loss of muscles . Dziki et al. have shown that the muscle regeneration by using scaffolds was histologically similar to that of uninjured muscle tissues. Moreover, the patients that were treated with ECM bioscaffolds demonstrated significant restoration of strength and functional improvement in addition to electromyographic improvement. In animal models, the use of ECM biologic scaffolds are associated with macrophage activation state transition in myogenic animals .

What these findings suggest is that the bioscaffolds that mimic the natural microenvironment of muscle tissues can be an attractive solution for the treatment of age-related muscle loss. Since they provide appropriate tissue-specific niche and direct macrophage response, the ECM scaffolds have the potential to alter the default response to skeletal muscle injury and hence provide an inductive template for facilitating muscle regeneration in the elderlies.

Classically, bioscaffolds were implanted by invasive surgical procedures. However, advancements have been made allowing for minimally invasive delivery of the bioactive components of these scaffolds in the target tissues. ECM hydrogels are one of these materials that can retain the composition and bioactivity of the intact natural ECM and can be delivered via syringes and catheters . Recently, matrix-bound nanovesicles have been identified within ECM bioscaffolds. These nanosized materials seem to be one of the chief signalling mechanisms for eliciting the biologic effect within the tissue. These matrix-bound nanovesicles also present a great potential to be used in a minimally invasive treatment strategy for sarcopenia.

[1]Barberi L, Scicchitano BM, De Rossi M, Bigot A, Duguez S, Wielgosik A, Stewart C, McPhee J, Conte M, Narici M, Franceschi C, Mouly V, Butler-Browne G, Musaro A: Age-dependent alteration in muscle regeneration: the critical role of tissue niche. Biogerontology 2013;14:273-292.

[2]Dziki J, Badylak S, Yabroudi M, Sicari B, Ambrosio A, Stearns K, Turner N, Wyse A, Boninger M, Brown E, Rubin J: An acellular biologic scaffold treatment for volumetric muscle loss: results of a 13-patient cohort Study. Nature Regenerative Medicine 2016

[3]Brown BN, Ratner BD, Goodman SB, Amar S, Badylak SF: Macrophage polarization: an opportunity for improved outcomes in biomaterials and regenerative medicine. Biomaterials 2012;33:3792-3802.

[4]Freytes DO, Martin J, Velankar SS, Lee AS, Badylak SF: Preparation and rheological characterization of a gel form of the porcine urinary bladder matrix. Biomaterials 2008;29:1630-1637.

Natural compounds to treat sarcopenia

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Jamila
Jamila Nov 25, 2020
Here are some examples of natural compounds that could treat sarcopenia:
  • In Chang's study, the researchers found that oligonol a compound commonly found in lychees could improve the grip strength and muscle mass of senescence‐accelerated mouse prone 8 mice.
  • Tomatidine is from green tomatoes (unripe tomatoes). Tomatidine supplementation was able to improve muscle mass and grip strength in aged mice.
  • Ryu and colleagues found that urolithin A was able to activate mitophagy in elderly mice. This led to an increase in muscle function. Urolithin is in berries, nuts, and pomegranates.

[1]Chang, Yun‐Ching, et al. "Oligonol alleviates sarcopenia by regulation of signaling pathways involved in protein turnover and mitochondrial quality." Molecular nutrition & food research 63.10 (2019): 1801102.

[2]Ebert, Scott M., et al. "Identification and small molecule inhibition of an activating transcription factor 4 (ATF4)-dependent pathway to age-related skeletal muscle weakness and atrophy." Journal of Biological Chemistry 290.42 (2015): 25497-25511.

[3]Ryu, Dongryeol, et al. "Urolithin A induces mitophagy and prolongs lifespan in C. elegans and increases muscle function in rodents." Nature medicine 22.8 (2016): 879-888.

Targeting the Senescent Associated Secretory Phenotype (SASP)

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Antonio Carusillo
Antonio Carusillo Nov 25, 2020
In my first contribution, I suggested the possibility of using CRISPR/Cas9 as an approach to address Sarcopenia by knocking-out the Myostatin gene.

However, we also have to consider that there could be some drawbacks which may pose concerns regarding the safety of the approach. Even if CRISPR is being used in clinical trials, ex vivo and in vivo, genetic engineering requires strict controls to avoid undesired side-effects.

For these reasons, I tried to look at other possibilities.

To better understand my next contribution, we have to zoom out a little bit.

Skeletal muscles are characterized by different progenitor cells, very important are the mesenchymal progenitor cells(MPCs). Their function is very important to support the myogenic cell ( meaning the “genesis” of muscle cells) function .

We also know from previous sessions that senescent cells are characterized by a permanent quiescent – no dividing – stage and feature a specific array of secreted molecules ( secretome) profile known senescence-associated secretory phenotype (SASP).

Why this information is important will be clarified in the following lines.

Skeletal muscles have a high regenerative potential upon stress and lesions introduced during physical effort or exercise. This is made possible by the presence of the so-called satellite cells which when a lesion is introduced they change their state from quiescence to proliferative, they fuse and form to new myotubes, the basic unit of muscle fibres
Muscle Progenitor Cells (MPCs) reside within the interstitial space of the skeletal muscle fibres and support the functionality of the satellite cells. Therefore, linking MPCs to muscle regeneration capability.
Since Sarcopenia is associated with a progressive skeletal muscle waste, very interesting was a recent paper were the MPCs have been investigated in the role of Sarcopenia .

In this particular study, a rat model used for ageing-related studies has been exploited to gain knowledge about what happens to MPCs in ageing skeletal muscles tissues. What it was observed was that such cells over-expressed the anti-proliferative genes p16 and p21, as well as accumulation of DNA damage, was detected, all these are well-established signatures of senescent cells .

Furthermore, these cells started secreting IL-6, TGFβ1, and CCL2 molecules which are part of the SASP
array .

These evidences strongly suggest that MPCs acquire a senescent phenotype.

To reveal the consequence of such change in the MSCs phenotype, senescent MSCs were cultured together with skeletal muscle primary cells. Via this strategy, the researchers observed that although the skeletal primary muscle cells could retain their proliferative state, they were unable to fuse, the key step in myotube formation.

Thus, hampering muscle regeneration.

This study, together with other pieces of evidence regarding the effect of senescent cells and SASP on the neighbouring cells , seems to point at the SASP itself as a possible target.

In particular, among the secreted molecules there is IL6. IL6 is a pleiotropic cytokine and can be released by inflammatory cells and by muscle fibres. At low levels/ transient levels, IL6 is important as it mediates satellite cells activation and proliferation. However, is the chronic expression by senescent cells has been associated with sarcopenia.
IL6 inhibition by blocking molecules has been showed to attenuate sarcopenia . We may envision an approach where systemic administration of IL6 inhibitors may help bettering the symptoms associated to Sarcopenia?
This is also supported by the fact that upon physical exercise skeletal muscle cells secrete IL-15 which attracts NK cells, which also are involved in the disposal of senescent cells . Therefore they play an important function in tissue repair and inflammation.
However, high IL-6 can inhibit NK cells recruitment .

This matches intriguingly with the observation that high IL-6 secretion is detrimental for skeletal muscle cells.
For these reasons, we may think about future studies aiming to better investigate if IL-6 inhibitors or other SASP inhibitors may ameliorate this phenotype.

Last, but not the least I would like to highlight again the role of MSCs in affecting muscle regeneration. In a previous session, we spoke about senolytics and among my contributions, there was the suggestion of turning to Chimeric Antigen Receptor (CAR) T cells. As you will find in the session, they have been successfully used not only to address blood malignancies but also to target senescent cells! So, we may think about developing anti-senescent-MSCs specific CART cells, to deplete the senescent MSCs. This may be a feasible approach, the challenge would be to identify a specific receptor able to target the CART cells only against the senescent MCSs and not against the “young” one. Further development of such a strategy would be to use NK cells and not T cells. NK cells, as reported above, are involved also in senescent cells disposal and have CAR-NK cells have been recently engineered . An anti-senescent cells CAR-NK cells may be even more effective than CAR T cells due to its natural tendency at getting ride of senescent cells!

[1]Klimczak A, Kozlowska U, Kurpisz M. Muscle Stem/Progenitor Cells and Mesenchymal Stem Cells of Bone Marrow Origin for Skeletal Muscle Regeneration in Muscular Dystrophies. Arch Immunol Ther Exp (Warsz). 2018;66(5):341-354. doi:10.1007/s00005-018-0509-7

[2]Yin H, Price F, Rudnicki MA. Satellite cells and the muscle stem cell niche. Physiol Rev. 2013;93(1):23-67. doi:10.1152/physrev.00043.2011

[3]Sugihara H, Teramoto N, Yamanouchi K, Matsuwaki T, Nishihara M. Oxidative stress-mediated senescence in mesenchymal progenitor cells causes the loss of their fibro/adipogenic potential and abrogates myoblast fusion. Aging (Albany NY). 2018;10(4):747-763. doi:10.18632/aging.101425

[4]Casella G, Munk R, Kim KM, Piao Y, De S, Abdelmohsen K, Gorospe M. Transcriptome signature of cellular senescence. Nucleic Acids Res. 2019 Aug 22;47(14):7294-7305. doi: 10.1093/nar/gkz555. Erratum in: Nucleic Acids Res. 2019 Dec 2;47(21):11476. PMID: 31251810; PMCID: PMC6698740.

[5]Coppé JP, Desprez PY, Krtolica A, Campisi J. The senescence-associated secretory phenotype: the dark side of tumor suppression. Annu Rev Pathol. 2010;5:99-118. doi:10.1146/annurev-pathol-121808-102144

[6]Marjolein P Baar, Eusebio Perdiguero, Pura Muñoz-Cánoves, Peter LJ de Keizer, Musculoskeletal senescence: a moving target ready to be eliminated, Current Opinion in Pharmacology, Volume 40, 2018, Pages 147-155, ISSN 1471-4892, https://doi.org/10.1016/j.coph.2018.05.007.

[7] 30. Tsujinaka T et al.: Interleukin 6 receptor antibody inhibits muscle atrophy and modulates proteolytic systems in interleukin 6 transgenic mice. J Clin Invest 1996, 97:244-249.

[8]O’Leary, M.F., Wallace, G.R., Bennett, A.J. et al. IL-15 promotes human myogenesis and mitigates the detrimental effects of TNFα on myotube development. Sci Rep 7, 12997 (2017). https://doi.org/10.1038/s41598-017-13479-w

[9]Kale A, Sharma A, Stolzing A, Desprez PY, Campisi J. Role of immune cells in the removal of deleterious senescent cells. Immun Ageing. 2020;17:16. Published 2020 Jun 3. doi:10.1186/s12979-020-00187-9

[10]Inhibition of Natural Killer Cell Cytotoxicity by Interleukin‐6: Implications for the Pathogenesis of Macrophage Activation Syndrome Loredana Cifaldi Giusi Prencipe Ivan Caiello Claudia Bracaglia Franco Locatelli Fabrizio De Benedetti Raffaele Strippoli

[11]Guozhu Xie, Han Dong, Yong Liang, James Dongjoo Ham, Romee Rizwan, Jianzhu Chen, CAR-NK cells: A promising cellular immunotherapy for cancer, EBioMedicine, Volume 59, 2020, 102975, ISSN 2352-3964, https://doi.org/10.1016/j.ebiom.2020.102975.

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