Journal Club

12.04.18 by

MISEV2018: Update to the Minimal Information for Studies of Extracellular Vesicles

Updated guidelines on Minimal Information for Studies of Extracellular Vesicles have now been published in the Journal of Extracellular Vesicles (JEV, Taylor & Francis) as MISEV2018.

The original MISEV2014 guidelines were released in 2014 by the Board of Directors of the International Society for Extracellular Vesicles (ISEV) to provide guidance in standardization of protocols and reporting in the EV field. Accumulating more than 800 citations since its release, the MISEV2014 guidelines have achieved the aim of becoming a guiding standard for researchers. A 2016 survey of ISEV members reaffirmed the need for guidelines and recommended that they be updated regularly…but with broad community input to accommodate and shape the quickly developing field.

MISEV2018 updates the topics of nomenclature, separation, characterization, and functional analysis, integrating the contributions of over 380 ISEV members, a strong tribute to the commitment of ISEV members. A two-page checklist summarizing the main points is also included.

So what’s new? MISEV2018 recommends the use of ‘extracellular vesicle’ as the preferred generic terminology for use in publications, in part due to challenges in confirming the biogenesis mechanisms of exosomes, microvesicles, and other particles, and in part due to the vague and varied uses of other terms. Separation and concentration options are now many and diverse; researchers should pick the methods most fit for downstream purpose and, more importantly, report these clearly and accurately. The EV-TRACK database (van Deun et al., Nature Methods, 2017) is supported as a means to record these details in order to improve clarity and reproducibility. To establish presence of EVs, examples of EV-enriched markers are provided, but the need for “negative” (better: “depleted”) markers is also highlighted. MISEV2018 adds topology as a recommended form of EV characterization, for example identifying where in or on a vesicle your favorite protein or RNA resides. It also recommends functional analysis of the ‘non-EV’ fractions to confirm EV-specific function (or not!). An appreciation of EV heterogeneity is included with a reminder that ‘larger EVs matter’ and a request to explore a range of EV subtypes in functional studies. Finally, although some of the specific details contained in MISEV2018 are focused on mammalian components, it is appreciated that the guidelines are applicable to non-mammalian and non-eukaryote research.

Please contact the corresponding authors, Clotilde Théry and Kenneth Witwer with any questions or comments.

For more information on the process of writing and publishing MISEV2018, see this white paper and Witwer et al., J. Extracell. Vesicles, 2017.

References

Lotvall J, Hill AF, Hochberg F, et al. Minimal experimental requirements for definition of extracellular vesicles and their functions: a position statement from the international society for extracellular vesicles. J. Extracell. Vesicles. (2014) 3: 26913. doi:10.1080/20013078.2018.1535750. PMID:25536934.

Witwer KW, Soekmadji C, Hill AF, et al. Updating the MISEV minimal requirements for extracellular vesicle studies: building bridges to reproducibility. J. Extracell. Vesicles. (2017) 6: 1396823. doi:10.1080/20013078.2017.1396823. PMID:29184626.

Théry C, Kenneth W Witwer KW, Aikawa E, et al. Minimal information for studies of extracellular vesicles 2018 (MISEV2018): a position statement of the International Society for Extracellular Vesicles and update of the MISEV2014 guidelines. J. Extracell. Vesicles. (2018) 7: 1535750. doi:10.1080/20013078.2018.1535750.

10.22.18 by

Cancer cells send out ‘drones’ to battle the immune system from afar


Image: Kelsey Burke

This post originated as a press release from the University of Pennsylvania.

Cancerous tumors are more than a lump of cells growing out of control; they participate in active combat with the immune system for their own survival. Being able to evade the immune system is indeed a hallmark of cancer. Now, researchers from the University of Pennsylvania show that, to assist in the fight, cancer cells release biological “drones,” small vesicles called exosomes circulating in the blood and armed with the protein PD-L1, which causes T cells to tire before they have a chance to reach the tumor and do battle.

The work, published in the journal Nature (Chen et al., 2018), is a collaboration between Wei Guo of Penn’s School of Arts and Sciences and Xiaowei Xu of the Perelman School of Medicine. While primarily focused on metastatic melanoma, the team found that breast and lung cancer also release the PD-L1-carrying exosomes.

The research offers a paradigm-shifting picture of how cancers take a systemic approach to suppressing the immune system. In addition, it also points to a new way to predict which cancer patients will respond to certain checkpoint inhibitor drugs, which disrupt immune suppression to fight tumors, and a means of tracking the effectiveness of such therapies.

“Immunotherapies are life-saving for many patients with metastatic melanoma, but about 70 percent of these patients don’t respond,” says Guo, a professor of biology. “These treatments are costly and have toxic side effects, so it would be very helpful to know which patients are going to respond. Identifying a biomarker in the bloodstream could potentially help make early predictions about which patients will respond and, later on, could offer patients and their doctors a way to monitor how well their treatment is working.”

“Exosomes are tiny lipid-encapsulated vesicles with the diameter less than one-hundredth of a red blood cell. What we have found with these circulating exosomes is truly remarkable,” says Xu. “We collected blood samples from melanoma patients treated with anti-PD1 checkpoint inhibitor therapy. This type of liquid biopsy assay allows us to monitor tumor-related immune suppression with time.”

One of the most successful innovations in cancer therapy has been the use of checkpoint inhibitor drugs, which are designed to block attempts by cancer cells to suppress the immune system to allow tumors to thrive and spread. One of the primary targets for this class of drugs is PD-1, a protein on the surface of T cells. Tumor cells express PD-L1, which interacts with PD-1, effectively turning off that cell’s anti-cancer response. Blocking that interaction using checkpoint inhibitors reinvigorates T cells, allowing them to unleash their cancer-killing power on the tumor.

While it was known that cancer cells carried PD-L1 on their surface, Guo, Xu and colleagues found, in the new work, that exosomes from human melanoma cells also carried PD-L1 on their surface. Exosomal PD-L1 can directly bind to and inhibit T cell functions. Identifying the exosomal PD-L1 secreted by tumor cells provides a major update to the immune checkpoint mechanism, and offers novel insight into tumor immune evasion.

“Essentially exosomes secreted by melanoma cells are immunosuppressive.” Guo says. “We propose a model in which these exosomes act like drones to fight against T cells in circulation, even before the T cells get near to the tumor.”

Since a single tumor cell is able to secrete many copies of exosomes, the interaction between the PD-L1 exosomes and T cells provides a systemic and highly effective means to suppress anti-tumor immunity in the whole body. This may help explain why cancer patients have weakened immune systems.

Because exosomes circulate in the bloodstream, they present an accessible way of monitoring the cancer-T cell battle through a blood test, compared to a traditional, more-invasive tumor biopsy. After an acute phase of treatment, the researchers envision such a test as a way to monitor how well the drugs are keeping cancer cells in check.

By measuring pre-treatment levels of PD-L1, oncologists may be able to predict the extent of tumor burden in a patient and associate that with treatment outcome. In addition, a blood test could measure the effectiveness of a treatment. For example, levels of exosomal PD-L1 could indicate the level of T cell invigoration by immune checkpoint inhibitors.

“In the future, I think we will begin to think about cancers as a chronic disease, like diabetes,” says Guo. “And just as diabetes patients use glucometers to measure their sugar levels, it’s possible that monitoring PD-L1 and other biomarkers on the circulating exosomes could be a way for clinicians and cancer patients to keep tabs on treatment. It’s another step toward precision and personalized medicine.”

 

Reference

Chen G. et al. Exosomal PD-L1 contributes to immunosuppression and is associated with anti-PD-1 response. Nature (2018) 560: 382–386. doi: 10.1038/s41586-018-0392-8 PMID: 30089911

Guo and Xu coauthored the work with Penn’s Gang Chen, Alexander C. Huang, Wei Zhang, Min Wu, Jiegang Yang, Beike Wang, Honghong Sun, Wenqun Zhong, Bin Wu, Xiaoming Liu, Lei Guan, Tin Li, Shujing Liu, Ruifeng Yang, Youtao Lu, Liyun Dong, Suzanne McGettigan, Ravi Radhakrishnan, Junhyong Kim, Youhai H. Chen, Giorgos C. Karakousis, Tara C. Gangadhar, Lynn M. Schuchter, and E. John Wherry, as well as collaborators from Wuhan University, The Wistar Institute, Xi’an Jiaotong University, the University of Texas MD Anderson Cancer Center, and the Mayo Clinic.

The research was supported by the National Institutes of Health (GM111128, GM085146, AI105343, AI108545, AI082630, and AI117950), Parker Institute for Cancer Immunotherapy, American Heart Association, Tara Miller Melanoma Foundation, University of Pennsylvania, Wistar Institute, Dr. Miriam and Sheldon G. Adelson Medical Research Foundation, CAST Foundation, and NSFC Foundation.

Wei Guo is a professor of biology in the School of Arts and Sciences, and Xiaowei Xu is a professor of pathology and laboratory medicine and of dermatology in the Perelman School of Medicine at the University of Pennsylvania.

Wei Guo, Xiaowei Xu, and Gang Chen are listed as inventors on a patient owned by the University of Pennsylvania related to this work. Guo and Xu serve on the Scientific Advisory Board and have equities in Exo Bio, a company that has licensed the patent from the University of Pennsylvania.

08.16.18 by , and

Directed migration: Cells navigate by extracellular vesicles

This blog was adapted and simplified from a Spotlight in the Journal of Cell Biology.

Directed movement along a chemical gradient – chemotaxis – is a fundamental behavior of cells in the body during processes like inflammation, embryogenesis, and cancer metastasis. While the intracellular signaling mechanisms underlying chemotaxis have been investigated by many groups, it is not well known how stable gradients of chemoattractant chemicals are generated and maintained outside the cell, especially given the rapid diffusion of chemicals after secretion.

The amoeba Dictyostelium discoideum (fondly known as Dicty) is a model organism often studied since its biology can shed light on that of human cells. Recent work from Kriebel et al. (2018) showed that extracellular vesicles (EVs) are central to the biology of chemotaxis in Dicty by demonstrating that the main chemical Dicty follows during chemotaxis is synthesized within and released from EVs.

Dictyostelia are soil-living amoebas that undergo chemotaxis toward cyclic adenosine monophosphate (cAMP) under starvation conditions. As cAMP is released from the rear of migrating cells, chemotaxis toward cAMP leads to aggregation of the amoebas into multicellular structures.

Kriebel et al. previously showed (2008) that the enzyme that synthesizes cAMP, adenylyl cyclase (ACA), is present in multivesicular bodies (MVBs) located at the rear of migrating amoebas. They have now shown that leader cells release ACA-enzyme-containing vesicular trails and that follower cells stream onto them in a head-to-tail fashion. The vesicular trails are highly chemotactic and direct the migration of the follower cells.

Measurement of ATP, the precursor used by the enzyme to synthesize the product cAMP, revealed that it is also present inside EVs. Narrowing down from the 68 transporter proteins found in Dicty, the researchers identified the one protein – ABCC8 – that transports cAMP from the inside to the outside of EVs in order to promote chemotaxis. Dicty amoebas with that transporter knocked out exhibit much less chemotaxis and streaming behavior, and fluorescently tagged ABCC8 reintroduced into those amoebas is visible in the vesicular trails but not the plasma membrane.

Altogether, these data indicate that the entire machinery for generating and releasing chemoattractants is contained within EVs: the catalytically active enzyme (ACA), its substrate (ATP), the product (cAMP), and the transporter (ABCC8; see figure).

EVs secreted from Dictyostelia synthesize and release cAMP to promote chemotaxis. cAMP is converted from ATP by enzyme ACA in EVs and secreted through the ABCC8 transporter. Secreted cAMP makes a gradient and promotes chemotaxis of follower cells.

Overall, Kriebel et al. (2018) describe an elegant system by which chemotactic signals are generated and sustained to promote streaming in a complex multicellular system, and they elucidate a major mechanism by which cells leave a memory of themselves. EVs are left in a breadcrumb-like trail behind cells and continue sending chemotactic signals to surrounding cells. The finding that EVs act as cell-independent entities to not only carry but also generate bioactive products is important because it shows that they can amplify signals.

The same group made similar findings previously for neutrophils, one of the main sentry cells of the immune system (Majumdar et al., 2016). In both cases, the presence in EVs of enzymes that can generate a chemical product continuously amplifies and sustains a signal beyond that which would occur if the EVs carried just the chemical. The findings in amoeba and human cells together suggest that amplification of chemotactic signals is an important function of EVs that is conserved across organisms and necessary to promote effective directional sensing.

In a different context, it has been reported that precursor miRNAs can be processed into mature miRNAs in exosomes in a cell-independent manner due to the presence of the RNA interference–silencing complex (RISC) machinery in breast cancer–associated exosomes (Melo et al., 2014) and that this activity is important to promote tumor aggressiveness. Along with Melo et al. (2014), Kriebel et al. (2018) provide direct evidence that EVs can act as an independent machinery to regulate biological processes such as chemotaxis or tumorigenesis via enzymatic activities.

References

Kriebel PW, Barr VA, Rericha EC, Zhang G & Parent CA . (2008) Collective cell migration requires vesicular trafficking for chemoattractant delivery at the trailing edge. J. Cell Biol. 183:949–961. doi: 10.1083/jcb.200808105

Kriebel PW, et al. (2018) Extracellular vesicles direct migration by synthesizing and releasing chemotactic signals. J. Cell Biol. doi: 10.1083/jcb.201710170

Majumdar R, Tavakoli Tameh A & Parent CA. (2016) Exosomes mediate LTB4 release during neutrophil chemotaxis. PLoS Biol. 14:e1002336. doi: 10.1371/journal.pbio.1002336

Melo, et al. 2014. Cancer exosomes perform cell-independent microRNA biogenesis and promote tumorigenesis. Cancer Cell 26:707–721. doi: 10.1016/j.ccell.2014.09.005

Sung BH & Weaver AM. (2018) Directed migration: Cells navigate by extracellular vesicles. J. Cell Biol. doi: 10.1083/jcb.201806018

07.05.18 by

Can circulating extracellular RNA provide clues to human aging?

When someone finds out that I work at the National Institute on Aging, they usually ask me “How can I stop aging?”. Although aging is inevitable, it is well-known that individuals age at different rates and that certain groups age more rapidly than others. Hence, one of our laboratory’s objectives is to identify why certain population groups age differently, with the goal to find biomarkers that can tell us an individual’s biological age (how old you seem) versus chronological age (the actual time you have been alive).

Several years ago, we began this journey by examining whether small non-coding RNAs called microRNAs (miRNAs) change with age. We focused first on these regulatory RNAs because data has shown that these RNAs are particularly stable in biofluids, such as serum and plasma, and can be identified readily using small RNA sequencing. We identified several serum miRNAs that change significantly with age in both humans and rhesus monkeys (Noren Hooten, Fitzpatrick, et al. 2013). In most cases, their abundance in circulation decreases as we get older. Interestingly, some of these miRNAs target and negatively regulate the expression of various inflammatory markers, suggesting that decreased expression of these circulating miRNAs may contribute to higher levels of inflammatory markers that have already been observed in the elderly.

More recently we have focused on establishing a more complete extracellular RNA (exRNA) profile of human aging. To do so, we developed a sequencing pipeline that enables us to sequence both small and long RNAs in one sequencing reaction, which lowers both the cost and labor required (Dluzen, Noren Hooten, et al. 2018). Cataloging what is the “normal” distribution of exRNA in young and old individuals and identifying age-dependent differences will aid in establishing important references for the study of age-related disease. The Ensembl database classifies RNA into various categories, termed biotypes. We found that most RNA biotypes were similar in distribution between young and old, but several biotypes, including mitochondrial tRNAs (Mt_tRNA), mitochondrial ribosomal RNAs (Mt_rRNA), and unprocessed pseudogenes, were significantly higher in older individuals.


Figure 1. Changes in circulating factors with age. A decrease in circulating levels of specific miRNAs occurs with age. On the other hand, unprocessed pseudogenes, Mt_tRNAs, and Mt_rRNAs increase in abundance with age.

Pathway analysis revealed that RNAs related to mitochondria, response to oxidative stress, and chromatin remodeling were all enriched in the circulation of older individuals, providing potential clues as to what pathways may be deregulated as humans age. We also further validated our sequencing results in a larger cohort of individuals and found age-related changes in a messenger RNA, a small nucleolar RNA, a pseudogene transcript, a small nuclear pseudogene transcript, and several additional miRNAs.

What was very interesting was that we identified many circular RNAs (circRNAs) in serum, which we have named ex-circRNAs. Recent attention has been focused on circRNAs, as this class of ncRNAs may be important modulators of gene expression. CircRNAs have long half-lives (i.e. are stable) compared to mRNAs, making them an attractive new serum biomarker. However, little is known about ex-circRNAs, and I anticipate that this will be an active area of interest in the coming years.

As we have begun to establish an exRNA profile of human aging, there remain important unanswered questions in the field. Currently, we do not fully understand how exRNA in the circulation reflects the health status of our cells and tissues. It has also proven difficult to ascertain which cell type is contributing exRNA into the circulation. Further research is needed to better understand these questions.

Our identification of changes in circulating miRNAs and exRNAs establishes baseline references for how these biomarkers change with human age. As the risk for many diseases including cancer, heart disease, and neurological diseases increase with age, it is important to consider age when examining these factors in relation to a specific disease. Although we have not identified the “fountain of youth” or the “magic elixir” for aging, we hope that establishing these profiles with normal aging will soon help to identify circulating biomarkers that can distinguish individuals with faster biological aging that may result in shortened health span and life span.

References
Dluzen DF, Noren Hooten N, De S, Wood H, Zhang Y, Becker KG, Zonderman AB, Tanaka T, Ferrucci L & Evans MK. Extracellular RNA profiles with human age. Aging Cell 2018;e12785. PMID 29797538.

Noren Hooten N, Fitzpatrick M, Wood WH, De S, Ejiogu N, Zhang Y, Mattison JA, Becker JG, Zonderman AB & Evans MK. Age-related changes in microRNA levels in serum. Aging (2013) 5: 725-740. PMID 24088671.

06.14.18 by

Exosomes containing amyloid beta protein propagate Alzheimer’s disease pathology

This post originated as a press release from Linköping University.

The waste-management system of the cell appears to play an important role in the spread of Alzheimer’s disease in the brain. A new study, published in the prestigious scientific journal Acta Neuropathologica, has focused on small membrane-covered droplets known as exosomes. It was long believed that the main task of exosomes was to help the cell to get rid of waste products. In simple terms, they were thought of as the cell’s rubbish bags. However, our understanding of exosomes has increased, and we now know that cells throughout the body use exosomes to transmit information. It’s now known that the exosomes can contain both proteins and genetic material, which other cells can absorb.

The Linköping researchers have shown in the new study that exosomes can also transport toxic aggregates of the protein amyloid beta, and in this way spread the disease to new neurons. Aggregated amyloid beta is one of the main findings in the brains of patients with Alzheimer’s disease, the other being aggregates of the protein tau. As time passes, they form ever-increasing deposits in the brain, which coincides with the death of nerve cells. The cognitive functions of a person with Alzheimer’s disease gradually deteriorate as new parts of the brain are affected.

“The spread of the disease follows the way in which parts of the brain are anatomically connected. It seems reasonable to assume that the disease is spread through the connections in the brain, and there has long been speculation about how this spread takes place at the cellular level,” says Martin Hallbeck, associate professor in the Department of Clinical and Experimental Medicine at Linköping University and senior consultant of clinical pathology at Linköping University Hospital.

Cells became diseased
In a collaboration with researchers at Uppsala University, he and his co-workers have investigated exosomes in brain tissue from deceased persons. The research team at Linköping University found more amyloid beta in exosomes from brains affected by Alzheimer’s disease than in healthy controls. Furthermore, the researchers purified exosomes from the brains from people with Alzheimer’s disease, and investigated whether they could be absorbed by cells cultured in the laboratory.

“Interestingly, exosomes from patients were absorbed by cultured neurons, and subsequently passed on to new cells. The cells that absorbed exosomes that contained amyloid beta became diseased,” says Dr. Hallbeck.

The researchers treated the cultured neurons with various substances that prevent exosomes from being formed, released, or absorbed by other cells. They were able to reduce the spread of the aggregated amyloid beta between cells by disrupting the mechanism in these ways. The methods used in these laboratory experiments are not yet suitable for treating patients, but the discovery is important in principle.

“Our study demonstrates that it is possible to influence this pathway, and possibly develop drugs that could prevent the spreading. The findings also open up the possibility of diagnosing Alzheimer’s disease in new ways, by measuring the exosomes,” says Martin Hallbeck.

The research has received financial support from donors that include the Swedish Research Council, the Swedish Alzheimer’s Foundation, and the Swedish Brain Foundation.

Sinha MS, Ansell-Schultz A, Civitelli L, Hildesjö C, Larsson M, Lannfelt L, Ingelsson M & Hallbeck M. Alzheimer disease pathology propagation by exosomes containing toxic amyloid-beta oligomers. Acta Neuropathologica AOP 13 June 2018. doi: 10.1007/s00401-018-1868-1

Translation by George Farrants.

05.18.18 by

An EVolving approach to directed enzyme prodrug therapy for cancer

This commentary originally appeared as an Editor’s Choice in Science Translational Medicine. Thanks to STM and Steven Jay for permission to reprint here.

The sci-fi thriller I, Robot tells the story of robots attempting to take over the world based on their interpretation of the three governing laws of their programming. This plan is thwarted with the help of Sonny, a unique robot who can ignore the three laws due to being programmed differently. This movie illustrates how selective programming can be a powerful tool that can be used to turn a subset of a population against the rest. This same concept underlies the strategy of gene-directed enzyme prodrug therapy (GDEPT) for cancer, which involves specific delivery of a gene to cancer cells that allows for subsequent activation of a systemically administered prodrug into a toxic form only in cells where an enzyme encoded by the delivered gene is present. Several GDEPT strategies have advanced to clinical trials; however, the specificity and fidelity of gene delivery are still limiting factors to successful translation.

Toward addressing these limitations, Wang et al. describe the use of modified extracellular vesicles (EVs) for targeted delivery of mRNA to cancer cells overexpressing the HER2 receptor. EVs are nanoscale vesicles secreted by many cell types that have been co-opted for a variety of therapeutic applications. However, targeted delivery using EVs has been challenging, as has encapsulation of large nucleic acid cargo. To address cargo encapsulation, the authors applied a transfection-based approach to successfully load exogenous mRNA encoding for the enzyme HChrR6 into EVs. To address targeting, the authors created a novel chimeric protein consisting of a HER2 antibody fragment to target the receptor on cancer cells and the C1C2 domain of lactadherin, which interacts with the EV membrane. By mixing mRNA-loaded EVs with purified chimeric protein, the EVs were endowed with targeting capability for HER2-overexpressing cancer cells. Delivery of these EVs followed by systemic administration of the prodrug 6-chloro-9-nitro-5-oxo-5H-benzo-(a)-phenoxazine (CNOB) resulted in near complete growth arrest of orthotopically implanted HER2-overexpressing breast tumors in mice.

This report establishes a new and versatile approach for improving GDEPT that could be applied to a wide variety of cancers and other diseases. Significant barriers to translation of this approach remain, most notably the problem of scalability of EV-based approaches. However, the methods and strategy described are likely to have broad utility in further developing both GDEPT and therapeutic EVs.

Highlighted Article
J.-H. Wang, A. V. Forterre, J. Zhao, D. O. Frimannsson, A. Delcayre, T. J. Antes, B. Efron, S. S. Jeffrey, M. D. Pegram, A. C. Matin, Anti-HER2 scFv-directed extracellular vesicle-mediated mRNA-based gene delivery inhibits growth of HER2-positive human breast tumor xenografts by prodrug activation. Mol. Cancer Ther. (2018) 17:1133-1142. PMID:29483213 doi:10.1158/1535-7163.MCT-17-0827

04.27.18 by

New tRNA sequencing method generates an atlas of human tRNAs

Despite being one of the earliest known classes of non-coding RNA molecules, transfer RNAs (tRNAs) are still notoriously difficult to study. The challenge is largely due to this molecule’s secondary structure, chemical modifications to its constituent nucleotides (see figure), and the multiplicity of tRNA genes. As the number of non-coding RNA datasets proliferates, it is becoming increasingly important for tRNA genes to be accurately annotated. In a recent study, Thomas Tuschl from Rockefeller University and colleagues tackled this problem by developing a new protocol for sequencing tRNAs. The new method enabled them to assemble an atlas of human tRNAs for other researchers to use in analyzing their non-coding RNA data.

Hydro-tRNA Sequencing
Transfer RNAs have thermodynamically stable secondary and tertiary structures, and their constituent nucleotides are highly modified by RNA editing. Both of these characteristics are problematic for traditional RNA sequencing methods. The key to the Tuschl lab’s protocol, called hydro-tRNA sequencing (hydro-tRNAseq), is a partial alkaline hydrolysis step that breaks the 60-100 nucleotide-long tRNA into smaller fragments with fewer RNA modifications. These fragments, 19-35 nucleotides in size, have weaker secondary structure and fewer RNA modifications per fragment than the parent tRNA.

Applying the method to short RNA extracted from human embryonic kidney (HEK293) cells resulted in an increase in the fraction of reads mapped to tRNA between 2% and 40%, depending on the depth of sequencing. The short fragment length also improved read accuracy per base compared to standard tRNA sequencing.

To develop a thorough and representative reference set of human tRNAs, the HEK293 dataset was subjected to iterative cycles of mapping to existing reference tRNAs followed by manual curation. In each round, all transcripts with an error distance (number of mismatches, insertions, and deletions) of 1-2 from a given tRNA were kept as candidate reference sequences if they could be attributed to a tRNA isoacceptor (i.e. a different tRNA that binds to the same amino acid). If not, assuming that other mismatches were caused by misidentifying a modified base, transcripts with more than 10% mismatches compared to reference were expanded into a set of all possible combinations of RNA modifications and included in the reference pool (see figure). This mapping and selection process was repeated until there were no longer any modified positions left with a mismatch frequency over 10% compared to reference.

Candidate pre-tRNA genes were obtained by mapping the final tRNA reference sequences back to the genome. Altogether, this analysis was able to account for 93% of the 114 million reads in the deepest library of HEK293 cells’ tRNAs.

tRNA Modification Sites

tRNA Modification Sites
The team identified sites of modification from the high frequency of mismatches during mapping caused by read errors there during reverse transcription. Here the reference nucleotide is at ring center, known modification outside the ring, and frequency of each nucleotide read at that site inside the ring.
Source: Cell Reports

The Added Power of SSB PAR-CLIP
Though hydro-tRNAseq greatly improved the reference dataset of human tRNAs, there was still a risk that it alone would miss pre-tRNAs expressed at low levels or processed quickly into mature tRNA. Previous efforts to assay that ephemeral population employed ChIP-seq of POLR3, the polymerase that transcribes all tRNA genes, but doing so assumed that polymerase binding always led to expression and complete processing. The Tuschl lab focused instead on SSB, a protein that binds to the 3′ end of pre-tRNAs, immunoprecipitating tRNAs crosslinked to SSB using a method called PAR-CLIP. As predicted, almost half of the reads from their SSB PAR-CLIP experiments mapped to pre-tRNAs. Combining SSB PAR-CLIP with hydro-tRNAseq allowed the team to better identify mature and pre-tRNAs with improved, accurate, nucleotide-level resolution.

This study supplies the community with several new and useful resources. Hydro-tRNAseq provides a new method to overcome many of the struggles of tRNA sequencing analyses. Combining this method with SSB PAR-CLIP enabled the construction of a comprehensive atlas of pre-tRNAs and mature tRNAs in humans. This methodology can now be applied to study the tRNA complement in other species to further dissect tRNA biology.

Reference
Tasos Gogakos T, Brown M, Garzia A, Meyer C, Hafner M, & Tuschl T. Characterizing Expression and Processing of Precursor and Mature Human tRNAs by Hydro-tRNAseq and PAR-CLIP. Cell Reports (2017) 20: 1463-1475. doi: 10.1016/j.celrep.2017.07.029

02.14.18 by

I ❤️ Exosomes

Our bodies are made up of trillions of cells that work together to keep us alive. A major challenge in their success is communication between cells in different parts of the body. Our cells have ingenious ways of overcoming this challenge, with exosomes emerging as key players. Exosomes are cell-derived vesicles that can carry cargo in the form of nucleic acids, lipids, or proteins from one cell to another. The sender cell packages cargo into an exosome, which then leaves the cell by being pinched off from the cell membrane. The exosome finds it way to a neighboring cell or into the bloodstream, from which it can be sent throughout the body. The exosome has signs on its surface that determine what cells can receive the cargo, so it only goes to the intended receiver. If a heart cell wants to talk to another heart cell, it puts markers on its exosomes that make them stick to other heart cells. When those exosomes are taken into the receiving cells, their cargo can bring about physiologic changes there.

Extracellular vesicles and their cargo. Source: BioProcess International.

Exosomes play a major role not only in our regular physiology but also in disease. One of the fields in which the role of exosomes is being uncovered is cardiovascular disease. For example, heart endothelial cells (cells that line the blood vessels) communicate with heart muscle cells via exosomes that contain microRNA, a kind of molecule that can decrease how many transcripts of a particular set of genes get made in the target cell. This process may play a role in the heart’s response to plaque formation. One can envision the possibility for engineering exosomes so that we can communicate with our bodies to treat or prevent disease. The lab of Dr. Susmita Sahoo at the Icahn School of Medicine at Mount Sinai is interested in doing just that.

Before talking about how Dr. Sahoo’s group is using exosomes in treating heart failure, let’s talk about a specific cause of heart failure: epitranscriptomics. You may or may not have heard of epigenetics, which is the study of heritable, chemical changes to DNA that do not change the sequence of the DNA. Epitranscriptomics is based on the exact same idea, but the change happens at the RNA level. One such epitranscriptomic modification is the addition or removal of methyl groups on adenosines within certain mRNAs in cells.

Structure of N6-Methyladenosine (m6A)

Heart muscle cells (cardiomyocytes) usually use electrical signals to interact and pulse in unison with a set rhythm. Work from Dr. Sahoo’s team suggests that decreasing levels of FTO, an enzyme that removes these methyl groups from RNA, leads to arrhythmia, a disturbance in that synchronized pulse. This finding is corroborated by the fact that failing hearts have low levels of FTO and elevated levels of mRNA methylation. Delivering exosomes with extra FTO to these cells might help them maintain healthy levels of FTO and decrease the chance of heart failure; this approach holds tremendous promise for the treatment of heart disease.

Dr. Sahoo’s research on exosomes is not limited to the failing heart. Recent work from her group suggests that a specific type of exosome, known to carry a marker called CD34 on its surface, improves angiogenesis, or formation of new blood vessels. Angiogenesis is a crucial step in healing after an injury. Dr. Sahoo’s group has shown that exosomes are able to improve healing in mice by providing microRNAs important for angiogenesis to cells near the site of injury. This work is not only important in helping patients after an injury, but it also teaches us about fundamental roles of microRNAs in angiogenesis and gene regulation.

We have outlined only some of the work going on in Dr. Sahoo’s lab. You can visit her website or watch her recent ERCC seminar to learn more about exosomes and her research on their role in cardiac medicine.

02.02.18 by

Optimizing the production of extracellular vesicles for therapeutic applications

Therapeutic exosomes and Huntington’s disease
Extracellular vesicles, specifically exosomes, are currently being explored as therapeutic delivery systems for disease-targeting RNA molecules. In a talk at the ERCC9 conference, Reka A. Haraszti, M.D., a researcher in Dr. Anastasia Khvorova’s group at the University of Massachusetts Medical School, described how exosomes could be used to treat Huntington’s disease, a progressive neurodegenerative disorder. There are currently no effective therapies for this illness, which is caused by a mutation in the Huntingtin gene. Exosomes capable of transporting molecular payloads designed to silence the defective Huntingtin gene represent a potential therapy for this fatal disease.

Comparison of exosome production methods
Technical challenges in the large-scale production of exosomes currently limit their utility for disease treatment. To address this issue, Dr. Haraszti and Dr. Khvorova’s group teamed up with MassBiologics to develop and compare two different exosome production methods for yield and therapeutic efficacy of the exosomes. They utilized Tangential Flow Filtration (TFF) and ultracentrifugation to isolate exosomes from the conditioned media of cultured mesenchymal stem cells.

In TFF, conditioned media is continuously swept along the surface of a filter while a downward pressure is applied to force molecules through the filter. This process is like shaking a sifter to concentrate large particles blocking the holes in the filter, allowing smaller particles to pass through. In contrast, ultracentrifugation works by placing the conditioned media in a column of viscous fluid and spinning rapidly to separate extracellular vesicles in the media by their differing densities.

The researchers found that isolation by TFF resulted in 10-100 times more exosomes than ultracentrifugation. TFF-generated exosomes were also more heterogenous and contained 10 times more protein.

Exosomes produced by TFF and ultracentrifugation were also studied for their therapeutic potential in Huntington’s disease. After purification, exosomes were loaded with small interfering RNA (siRNA) molecules that could silence the expression of the mutant Huntingtin gene in target cells that take up the exosomes.

In cell cultures of primary neurons, TFF-generated exosomes showed greater inhibition of Huntingtin expression than those generated by ultracentrifugation. Moreover, TFF-generated exosomes infused into the brains of mice suppressed Huntingtin gene expression in vivo.

Future for therapies using exosomes isolated by Tangential Flow Filtration
The findings of Dr. Haraszti’s group indicate that Tangential Flow Filtration can generate a higher yield of exosomes for clinical use than older methods. Further, TFF-generated exosomes were effective gene therapy agents in experimental models of Huntington’s disease, and hold promise as delivery systems for clinical treatments.
 
 
Related Mini-conference

There is an upcoming mini-conference on EV manufacturing and isolation, a topic closely related to the research described here. The in-person conference is in Gainesville, Florida, but a webcast will also be available for those who want to participate remotely.
 
 
References
1. Haraszti RA, et al. Loading of extracellular vesicles with chemically stabilized hydrophobic siRNAs for the treatment of disease in the central nervous system. Bio-protocol (2017) 7: e2338. doi: 10.21769/BioProtoc.2338
2. Schwartz L., and Seeley K. Introduction to tangential flow filtration for laboratory and process development applications. Retrieved from https://laboratory.pall.com/content/dam/pall/laboratory/literature-library/non-gated/id-34212.pdf
3. Sunkara V, Woo HK, and Cho YK. Emerging techniques in the isolation and characterization of extracellular vesicles and their roles in cancer diagnostics and prognostics. Analyst (2016) 141: 371-81. doi: 10.1039/c5an01775k
4. Synder Filtration. “Characterization of polymeric, porous membranes: UF/MF & solute rejection measurements.” Retrieved from https://synderfiltration.com/learning-center/articles/membranes/characterization-of-polymeric-porous-membranes/

01.19.18 by

The landscape of RNA in different extracellular complexes: where should we look for biomarkers?

A recent study by Wei et al., 2017 catalogs the composition and characteristics of extracellular RNA (exRNA) secreted via three different routes from parent cells. The work provides novel insights into the biology of exRNA transport and intercellular communication, as well as the clinical potential of exRNA as a biomarker of disease.

The senior investigator of the study, Anna Krichevsky, Ph.D., at Brigham and Women’s Hospital and Harvard Medical School described the rationale for the study: “To understand the functions of exRNA complexes, we first have to define the exRNA repertoire with minimal bias.”

The exRNA Composition of Microvesicles, Exosomes, and Ribonucleoproteins (RNPs)
Dr. Krichevsky’s group sequenced RNA in microvesicles, exosomes, and extravesicular ribonucleoproteins (RNPs) isolated from glioma stem cells. They found that the majority of exRNA in all three fractions is noncoding. Although most exRNA studies focus on one class of noncoding RNA called microRNA (miRNA; 21-25 nucleotide molecules that repress gene expression), they reported that <10% of exRNA secreted by glioma stem cells is miRNA.

Comparing the profile of exRNA isolated from RNPs and extracellular vesicles (EVs) — including both exosomes and microvesicles, they found that RNPs contain higher amounts of noncoding cytoplasmic Y RNA and transfer RNA (tRNA) fragments.

Y RNA folds into a characteristic stem-loop structure and was originally found in protein-RNA complexes of individuals with autoimmune diseases. According to Dr. Krichevsky, “Despite abundant expression in all vertebrate cells, the physiological functions of Y RNA are only beginning to emerge.” Some evidence suggests that Y RNA plays a role in DNA replication and RNA quality control. Y RNA fragments may also be involved in cell death, ribosomal RNA maintenance, and histone gene expression.

tRNA is well known as a mediator of the translation of mRNA to protein. However, recent studies suggest that tRNA fragments found in exRNA are involved in regulating gene expression during cellular stress responses. Dr. Krichevsky discussed the implications of extracellular tRNA on cellular communication: “Based on the exRNA levels and biological functions of tRNA, we hypothesize that transferred tRNA transcripts can have a major impact on recipient cells.”

Along with noncoding RNA, a small proportion of gene-encoding messenger RNA (mRNA) was detected in extracellular vesicles and RNPs. Previous studies have also found extracellular mRNA; however, they did not determine if the mRNA transcripts were intact or fragmented. Dr. Krichevsky’s group was the first to show that short (<1000 nucleotides), endogenous full-length mRNAs can be packaged into exosomes, and microvesicles contain even longer mRNAs. Fragments of long mRNA transcripts were also present in exRNA.

Using exRNA as a Biomarker
Researchers are currently exploring the use of exRNAs as potential biomarkers for the diagnosis and monitoring of diseases. However, many exRNA biomarker studies have been limited in scope because they examined a heterogeneous pool of exRNA purified from an unfractionated collection of EV types.

By fractionating conditioned media from glioma stem cells into microvesicle, exosome, and RNP fractions, Dr. Krichevsky’s group was able to compare their RNA profiles with those of the parent cells. They found that the RNA content of microvesicles most closely resembled that of the parent cells, making this type of exRNA carrier a good candidate for disease biomarkers.

Dr. Krichevsky said, “We believe there is more intact mRNA in microvesicles, which we can consider for biomarkers. We can think about genes that are mutated. On the other hand, miRNAs are more enriched in the exosomes. It would be great if we could detect cancer mutations and non-coding RNA biomarkers in biofluids; then we would not need to do a biopsy.”

Conclusions
By developing novel experimental approaches to illustrate that exRNA composition differs by exRNA carrier, Krichevsky’s group has made significant contributions to exRNA research. Moreover, they highlighted that exRNA contains more than the well-studied miRNAs, including full-length mRNA molecules, Y RNA, and tRNA. Their data also indicate that the RNA profile of microvesicles is most similar to that of the cell of origin, including the presence of full-length mRNAs, making microvesicle exRNA a good candidate for some disease biomarkers.

In an interview, Dr. Krichevsky discussed the importance of this study: “Our work changed the way people thought about exRNA by showing them the exRNA in numbers, which helps appreciate their heterogeneity and the overall impact. The field is shifting from focusing on a specific extracellular miRNA to now considering that there are thousands of different RNAs present in extracellular complexes.”

References
Wei, Z. et al. Coding and noncoding landscape of extracellular RNA released by human glioma stem cells. Nature Communications (2017) 8:1145. doi:10.1038/s41467-017-01196-x

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