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Developing therapeutic microRNAs for cancer
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Micrornas As Tools In Biopharmaceutical Production
You have support affirms not crack! You are refugee has not suit! The climate remains then believed. Gabriela Nicolescu, Pieter J. Arno Scherzberg, Joachim H.
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Benjamin Libet, Professor Stephen M. Additionally, exosomes efficiently deliver their content through cellular membranes and can be modified to ensure targeted transport [ 9 , 43 , 44 ]. Different immortalized cell lines have commonly been used as producers for extracellular vesicles owing to the infinite supply of cells for production, increased proliferation rates and the ease of genetic modification [ 31 , 45 ]. However, most immortalized cell lines originate from tumorigenic origin which eliminates their use for therapeutic exosome production since they may carry harmful or even carcinogenic constituents [ 46 ].
As an alternative, mesenchymal stem cells MSCs have been used for exosome production and already showed promising results in several approaches [ 47 — 49 ]. Nevertheless, MSCs are not ideal for exosome production, as large-scale cultivation is difficult and the final yield remains unsatisfactory [ 50 ]. In this study we evaluated the potential of the human amniocyte derived CAP cell line to be used for exosomes production.
CAP cells are from human, non-tumorigenic origin and hold a fully documented history, since they were initially developed for the production of therapeutic proteins and viral vectors [ 32 , 33 ]. In addition, their ability to grow in suspension may greatly simplify future production and purification of exosomes at a larger scale. Taking together, CAP cells have significant advantages over currently used cell lines. For the initial evaluation of CAP cells as production hosts for exosomes, a parental cell line was engineered to overexpress the exosome marker protein CD63 fused to GFP. This allowed for successful tracing of produced exosomes and validation of their isolation using FACS analysis, as well as tracing their delivery to recipient cells using fluorescence microscopy.
In contrast to cellular preloading, exosomes also have been loaded with miRNAs after their isolation through electroporation [ 9 , 51 ]. However, this approach not only carries the risk for contamination with transfection reagents, but also shows limited transfection efficiencies [ 52 ]. Furthermore, the morphology of exosomes is altered by electroporation, leading to adverse effects such as aggregation [ 53 ]. With this study we aimed to explore, if CAP cells are capable of producing functional exosomes loaded with efficient levels of therapeutic miRNAs, which can be used for miRNA delivery to recipient cells.
Detailed characterization of CAP exosomes isolated from culture medium via several experiments verified the presence of membrane surrounded vesicles of 50— nm diameter, displaying the exosome marker protein CD9 as well as CDGFP on their membrane Fig 1. Furthermore, these vesicles contained mature miR and miR, which were successfully pre-loaded into exosomes by either cellular pathways or passive integration during exosome biogenesis Fig 2. These data indicate that CAP cells are not just able to produce exosomes but are also capable of efficiently loading those exosomes with therapeutic miRNAs.
While, miRNA promotes apoptosis in liver cancer and suppresses tumorigenesis in hepatocellular carcinoma [ 56 , 57 ], miRNA is reported to induce apoptosis in ovarian and cervical carcinoma [ 40 , 58 ], but is contradictory known to enhance cancer progression and metastasis in laryngeal squamous cell carcinoma [ 59 ].
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Due to these impacts on cell growth, negative effects of their overexpression on the CAP host cells may limit the potential of exosomal pre-loading strategies. Human amniocytes, where CAP cells were originally derived from, have previously been shown to possess stem-cell-like characteristics and express canonical regulators associated with pluripotency and stem cell repression [ 60 ], which may render them with a higher resistance to inducers of apoptosis [ 61 , 62 ]. Alternatively, relevant target genes of pro-apoptotic miRNAs in tumor cells may be absent in amniocytes of non-tumorigenic origin and thereby enable overexpression in an efficient manner.
The cell line-specific mechanism of miRNAs, has already been observed for miRNA, which showed both pro-apoptotic and anti-apoptotic effects in two different tumorigenic cell lines [ 59 , 63 ]. It is therefore not surprising that miRNA and miRNA exhibit different effects in cell lines derived from amniocytes and ovarian cancer, respectively. Importantly, this cellular property of resistance to some pro-apoptotic miRNAs may represent an advantage for the production process of exosomes containing therapeutic molecules and render CAP cells as an ideal host for exosome production. After accessing efficient production and loading of CAP exosomes with therapeutic miRNAs, their functionality was investigated.
Exosomes interact with target cells via membrane fusion, endocytosis or interaction with membrane receptors [ 8 , 64 , 65 ]. However, no co-localization with endosomes or lysosomes was observed, which corresponds to similar findings describing exosomes uptake mainly by endocytosis into the cytoplasm using exosomes from tumorigenic origin [ 65 , 66 ].
Since miRNA regulation of these target genes was reported to induce apoptosis in ovarian cancer cells, their downregulation after exosomal delivery of miRNA verifies the functionality and potency of exosomes to transport therapeutic molecules to target cells.
MicroRNAs as Tools in Biopharmaceutical Production
In addition, RAF1, which is also known as the target gene of miRNA, shows a clear trend towards downregulation, however, significane was not reached. In contrast to our study, Kleemann et al. These high levels were probably not achieved using cellular loading of exosomes [ 39 ].
Nethertheless, the exact mechanisms of exosomal miRNA loading and release are not fully understood and require further investigation. In summary, this study describes human amniocyte derived CAP cells as a novel source for engineered exosomes of non-tumorigenic origin. Furthermore, it was demonstrated that pro-apoptotic miRNAs could be pre-loaded via cellular pathways into exosomes. Most importantly, exosomes produced by CAP cells delivered functional pro-apoptotic miRNAs to receptor cells, supporting their potential to produce exosomal delivery vehicles for therapeutics.
- Human CAP cells represent a novel source for functional, miRNA-loaded exosome production.
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By revealing CAP cells as a novel and efficient cell line for exosome production, the pharmaceutical use of exosomes could become strikingly more feasible. Passaging to a maximum of 20 passages was performed every 3—4 days to a denstiy of 0. For growth evaluation of engineered CAP-cells, they were seeded in a working volume of 50 ml with a density of 0. Viable cell density and viability were measured over a period of 7 days. Cell suspension was centrifuged for 15 min at RT with x g to pellet the cells.
Afterwards, the supernatant was 0.