Project Description

"Equpped with his five senses, man explores the universe around him and calls the adventure Science" Edwin Powell Hubble

The challenge

As a result of late diagnosis and limited treatment options, many malignant tumors, such as lymphomes, have a poor prognosis and require more innovative approaches to eradicate. Burkitt’s lymphoma (BL) is a rapidly proliferating lymphoma of germinal-center B cells. Given the rarity of BL and the paucity of clinical trials, the optimal therapeutic approach is controversial, particularly in adults in whom toxicity is a significant challenge with many regimens.

The problem

With the development of biomedicine, the use of extracellular vesicles, such as exosomes (EXOs) is rapidly developing as a new therapy for tumors. As biological carriers, EXOs possess biological activity and can transport their contents between cells. The contents are natural or artificially loaded with biomolecules or chemical drugs. However, the treatments of tumors through the delivery of EXOs are not sufficiently accurate or efficient, and various challenges need to be overcome. In the future, for the promotion and application of exosomes, it is of great significance to understand how to select appropriate exosomes loaded with biomolecules or chemical drugs for different tumors types, and how to deliver exosomes to recipient cells accurately and efficiently.


Detailed explanation of all the problems

The clinical importance of EXOs has been established in their use as alternatives to liposome-mediated drug delivery in cancer immunotherapy. EXOs are also a promising biological gene delivery system due to their microRNA and mRNA content. (2,4)

EXOs have been previously studied and described mainly in Mesenchymal Stem Cells (MSC), as they are a natural producer of these nanovesicles. Although the study of MSC-exosomes has achieved a lot of promising results, in general, our exploration in the field of EXOs is still in its infancy (5). At present, there are still some problems to which we need to pay attention. For example, the methods of exosomal isolation and purification are not uniform, which affects the reproducibility of research results. The fate of EXOs after entering the recipient cells (targeting), the specificity of organ distribution, and the treatment mechanism of disease are still unclear, as well as efficient methods for cargo loading into EXOs (6). In addition to these obstacles, low yield (7), heterogeneity, difficulty preserving, and so on need to be solved urgently (8). It has also been difficult to achieve standardized, large-scale production.

In order to give a practical application to EVs, isolation and purification techniques of EXOs ought to be established and protocolized. Until now, isolation of EVs subpopulation has been done by combination of various expensive methods such as Differential Ultracentrifugation and Density Gradient Flotation, Size Exclusion Cromatography (SEC), Ultrafiltration or Immunocapture (9,10). However, membrane damage of exosomes may occur during centrifugation. Additionally, ultracentrifugation requires long time and expensive equipment support (11). The characteristic of SEC is time-saving, low cost, and good repeatability, but the recovery rate and purity of EXOs are reduced. So, there is clearly no consensus as to which was the most efficient technique that could purify EV subpopulations without being so expensive. In this paper, we review a His-tag affinity chromatography to establish once and for all isolation protocols.

Another important limitation is the production of EXOs itself. The urge of EVs demand for clinical approaches requires the development of standard, scalable, and cost-effective approaches for their production. For EXOs-based therapeutics, manufacturing requires high capacity and scalability without influencing the composition or potency of EXOs (12). Until this day, production and isolation of EXOs has been mainly done using Mesenchymal Stem Cells (MSC). MSC have proven to have limited expansion capability, and undergo senescence after a few passages, and EXOs derived from senescent stem cells have impaired regenerative capacity compared to young cells (13). MSCs also present difficulties in order to be extracted and cultivated, compared to other cell lines.

Our solution

In order to overcome these drawbacks, we propose a new system for the production and dosage of EXOs. We report a transfected HEK293 cell line enhanced for exosomal synthesis, enabling efficient and scalable production, overexpressing genes involved in EXOs biogenesis. We also modified the exosomal surface membrane, allowing a specific interaction with the target cells we aim to deliver the exosomal cargo. Furthermore, we aim to load our EXOs with an anti-c-myc siRNA with help of Archeal RNA Binding Protein (RBP), L7Ae conjugated to a CD63-tag on the exosomal membrane. Purification and isolation of EXOs will be done via nickel affinity cromatography using Ni-NTA. To conclude, our functional validation will be assessed targeting our EXOs to B lymphocytes overexpressing c-myc oncogene in Burkitt’s Lymphoma.


Detailed description

Extracellular vesicles (EVs) are a heterogeneous group of small, lipid-based nanoparticles that are delivered by almost all cell types and play a key role as mediators of many physiological and pathophysiological processes. EVs can be classified according to their origin, function, cargo or biogenesis (1), but we can divide them into three main groups: exosomes (hereinafter referred to as EXOs), microvesicles, and apoptotic bodies. EXOs comprise a small fraction of EVs, which are produced by all types of cells and are secreted into the extracellular environment. EXOs originate from multivesicular bodies (MVB), originating from outward budding at the plasma membrane, with EXOs displaying the exosomal markers CD63, CD81, and CD9, derived from MVB-plasma membrane fusion events. (2,3).

The clinical importance of EXOs has been established in their use as alternatives to liposome-mediated drug delivery in cancer immunotherapy. EXOs are also a promising biological gene delivery system due to their microRNA and mRNA content. (2,4)

EXOs have been previously studied and described mainly in Mesenchymal Stem Cells (MSC), as they are a natural producer of these nanovesicles. Although the study of MSC-exosomes has achieved a lot of promising results, in general, our exploration in the field of EXOs is still in its infancy (5). At present, there are still some problems to which we need to pay attention. For example, the methods of exosomal isolation and purification are not uniform, which affects the reproducibility of research results. The fate of EXOs after entering the recipient cells (targeting), the specificity of organ distribution, and the treatment mechanism of disease are still unclear, as well as efficient methods for cargo loading into EXOs (6). In addition to these obstacles, low yield (7), heterogeneity, difficulty preserving, and so on need to be solved urgently (8). It has also been difficult to achieve standardized, large-scale production.

In order to give a practical application to EVs, isolation and purification techniques of EXOs ought to be established and protocolized. Until now, isolation of EVs subpopulation has been done by combination of various expensive methods such as Differential Ultracentrifugation and Density Gradient Flotation, Size Exclusion Cromatography (SEC), Ultrafiltration or Immunocapture (9,10). However, membrane damage of exosomes may occur during centrifugation. Additionally, ultracentrifugation requires long time and expensive equipment support (11). The characteristic of SEC is time-saving, low cost, and good repeatability, but the recovery rate and purity of EXOs are reduced. So, there is clearly no consensus as to which was the most efficient technique that could purify EV subpopulations without being so expensive. In this paper, we review a His-tag affinity chromatography to establish once and for all isolation protocols.

Another important limitation is the production of EXOs itself. The urge of EVs demand for clinical approaches requires the development of standard, scalable, and cost-effective approaches for their production. For EXOs-based therapeutics, manufacturing requires high capacity and scalability without influencing the composition or potency of EXOs (12). Until this day, production and isolation of EXOs has been mainly done using Mesenchymal Stem Cells (MSC). MSC have proven to have limited expansion capability, and undergo senescence after a few passages, and EXOs derived from senescent stem cells have impaired regenerative capacity compared to young cells (13). MSCs also present difficulties in order to be extracted and cultivated, compared to other cell lines.

In order to overcome these drawbacks, we propose a new system for the production and dosage of EXOs. We report a transfected HEK293 cell line enhanced for exosomal synthesis, enabling efficient and scalable production, overexpressing genes involved in EXOs biogenesis. We also modified the exosomal surface membrane, allowing a specific interaction with the target cells we aim to deliver the exosomal cargo. Furthermore, we aim to load our EXOs with an anti-c-myc siRNA with help of Archeal RNA Binding Protein (RBP), L7Ae conjugated to our CD63-tag on the exosomal membrane. To conclude, our functional validation will be assessed targeting our EXOs to B lymphocytes overexpressing c-myc oncogene in Burkitt’s Lymphoma.

Bibliography

  1. Veziroglu, E. M., & Mias, G. I. (2020). Characterizing extracellular vesicles and their diverse RNA contents. Frontiers in Genetics, 11, 700. DOI:https://doi.org/10.3389/fgene.2020.00700
  2. Bunggulawa E.J., Wang W., Yin T., Wang N., Durkan C., Wang Y., Wang G. Recent advancements in the use of exosomes as drug delivery systems 06 biological sciences 0601 biochemistry and cell biology. J. Nanobiotechnol. 2018;16:81. DOI:https://doi.org/10.1186/s12951-018-0403-9
  3. Peng, X., Yang, L., Ma, Y. et al. Focus on the morphogenesis, fate and the role in tumor progression of multivesicular bodies. Cell Commun Signal 18, 122 (2020). DOI:https://doi.org/10.1186/s12964-020-00619-5
  4. O’Loughlin C.A.W.A.M.J.W.A.J., Woffindale C., Wood M. Exosomes and the Emerging Field of Exosome-Based Gene Therapy. Curr. Gene Ther. 2012;12:262–274 DOI: https://doi.org/10.2174/156652312802083594
  5. Wei W, Ao Q, Wang X, Cao Y, Liu Y, Zheng SG, Tian X. Mesenchymal Stem Cell-Derived Exosomes: A Promising Biological Tool in Nanomedicine. Front Pharmacol. 2021 Jan 25;11:590470. https://doi.org/10.3389/fphar.2020.590470
  6. Li, Sp., Lin, Zx., Jiang, Xy. et al. Exosomal cargo-loading and synthetic exosome-mimics as potential therapeutic tools. Acta Pharmacol Sin 39, 542–551 (2018). https://doi.org/10.1038/aps.2017.178
  7. Shao, L., Zhang, Y., Pan, X., Liu, B., Liang, C., Zhang, Y., et al. (2019). Knockout of beta-2 microglobulin enhances cardiac repair by modulating exosome imprinting and inhibiting stem cell-induced immune rejection. Cell. Mol. Life Sci. 77 (5), 937–952. https://doi.org/10.1007/s00018-019-03220-3
  8. Pegtel, D. M., and Gould, S. J. (2019). Exosomes. Annu. Rev. Biochem. 88, 487–514. https://doi.org/10.1146/annurev-biochem-013118-111902
  9. Théry C, Amigorena S, Raposo G, Clayton A. Isolation and characterization of exosomes from cell culture supernatants and biological fluids. Curr Protoc Cell Biol (2006) 3:3.22.DOI:https://doi.org/10.1002/0471143030.cb0322s30
  10. Willms E, Cabañas C, Mäger I, Wood MJA and Vader P (2018) Extracellular Vesicle Heterogeneity: Subpopulations, Isolation Techniques, and Diverse Functions in Cancer Progression. Front. Immunol. 9:738. DOI:https://doi.org/10.3389/fimmu.2018.00738
  11. Linares R, Tan S, Gounou C, Arraud N, Brisson AR. High-speed centrifugation induces aggregation of extracellular vesicles. J Extracell Vesicles. 2015;4:29509. DOI:https://doi.org/10.3402/jev.v4.29509
  12. Whitford, William; Guterstam, Peter (2019). Exosome manufacturing status. Future Medicinal Chemistry, 11(10), 1225–1236. DOI:https://doi.org/10.4155/fmc-2018-0417
  13. Chen T.S., Arslan F., Yin Y., Tan S.S., Lai R.C., Choo A.B.H., Padmanabhan J., Lee C.N., de Kleijn D., Lim S.K. Enabling a robust scalable manufacturing process for therapeutic exosomes through oncogenic immortalization of human ESC-derived MSCs. J. Transl. Med. 2011;9:47. DOI:https://doi.org/10.1186/1479-5876-9-47
  14. Liang Y, Duan L, Lu J, Xia J. Engineering exosomes for targeted drug delivery. Theranostics. 2021 Jan 1;11(7):3183-3195. DOI:https://doi.org/ 10.7150/thno.52570
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