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Adipocytes Provide A Source Of Exosome-packaged Mirna For Target Cell Uptake
Ziyu Chen, MD, PhD, Mengfan Wu, MD, PhD, Shailesh Agarwal, MD.
Harvard Medical School, Boston, MA, USA.

PURPOSE: MicroRNA-based therapy presents an enormous clinical opportunity. However, their translation has been limited due to shortened half-life and poor bioavailability. Strategies involving liposomal or nanoparticle-based miRNA delivery have sought to address these challenges by protecting miRNA from serum RNases and enabling uptake through the non-polar lipid membrane. However, these strategies remain poor options due to the need for frequent re-dosing and the absence of an endogenous source of long-term miRNA delivery. We hypothesize that adipocytes derived from adipose tissue can be modified to secrete exosome-packaged miRNA for uptake in a target cell population.
METHODS: Lentivectors encoding miR-122-5p with XMotif exosome-packaging tag (System Biosciences) were transfected into HEK293 cells via Lipofectamine 3000 at a final concentration of 2.5ug/ml, or into human adipocytes via electroporation (50 ug lentivector in 200ul adipocytes and 200ul PBS, 500V/5msec/4 pulses) yielding HEK293/miR-122-5p or Adipocyte/miR-122-5p. Control cells were transfected without lentivectors. Following transfection, cells were cultured in DMEM/1% pen-strep for 48 hours. Exosomes were then isolated from culture media (Exo-Quick TC, System Biosciences). Separately, HEK293 or AsPC1 cells were transfected with the LightSwitch RIMS1 3’UTR reporter vector (Active Motif) via Lipofectamine 3000. Resulting HEK293/3’UTR-luciferase or AsPC1/3’UTR-luciferase reporters were treated with purified exosomes from HEK293 (positive control) or adipocytes (final concentration 25 ug/mL) (n=6). After 24 hours, luciferase assay of the reporter cells was performed (LightSwitch luciferase assay kit, Active Motif) to test bioactivity.
RESULTS: Exosome concentrations in the culture media after 48h incubation was quantified (HEK293/control: 1.9ug/ml, HEK293/miR-122-5p: 3.19ug/ml, adipocyte/control: 1.2ug/ml, adipocyte/miR-122-5p: 2.1ug/ml). Luciferase signal significantly decreased in HEK293/3’UTR-luciferase or AsPC1/3’UTR-luciferase reporter cells treated with HEK293/miR-122 exosomes relative to respective cells treated with control exosomes (HEK293/3’UTR-luciferase: 0.52 v. 1, p<0.05 and AsPC1/3’UTR-luciferase: 0.69 v. 1, p<0.05) (Fig A/C). Similarly, luciferase signal significantly decreased in HEK293/3’UTR-luciferase or AsPC1/3’UTR-luciferase reporter cells treated with Adipocyte/miR-122 exosomes relative to respective cells treated with control exosomes (HEK293/3’UTR-luciferase: 0.66 v. 1.0, p<0.05 and AsPC1/3’UTR-luciferase: 0.29 v. 1.0, p<0.001) (Fig B/D).
CONCLUSION: Our results showed that human adipocytes can serve as a cell therapy capable of secreting a pre-specified exosome-packaged miRNA. Importantly, these findings demonstrate that these exosomes can be taken up by different cell types (HEK293 and AsPC1 pancreatic cancer cells). Finally, not only are these exosomes taken up, but their contents are capable of exerting a desired biologic effect (e.g. reporter downregulation). Our findings provide strong support for a cell therapy which provides long-term endogenous exosome-packaged miRNA secretion. Future near-term studies will quantify exosome miRNA contents using qRT-PCR and verify durable secretion with the lentivector system. Long-term studies will deploy this technology using miRNAs targeted to improve wound healing disorders and malignancy.


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