Generation and testing of clinical-grade exosomes for pancreatic cancer
Mayela Mendt, Sushrut Kamerkar, Hikaru Sugimoto, Kathleen M. McAndrews, Chia-Chin Wu, Mihai Gagea, Sujuan Yang, Elena V. Rodriges Blanko, Qian Peng, Xiaoyan Ma, Joseph R. Marszalek, Anirban Maitra, Cassian Yee, Katayoun Rezvani, Elizabeth Shpall, Valerie S. LeBleu, Raghu Kalluri
Mayela Mendt, Sushrut Kamerkar, Hikaru Sugimoto, Kathleen M. McAndrews, Chia-Chin Wu, Mihai Gagea, Sujuan Yang, Elena V. Rodriges Blanko, Qian Peng, Xiaoyan Ma, Joseph R. Marszalek, Anirban Maitra, Cassian Yee, Katayoun Rezvani, Elizabeth Shpall, Valerie S. LeBleu, Raghu Kalluri
View: Text | PDF
Research Article Oncology

Generation and testing of clinical-grade exosomes for pancreatic cancer

Abstract

Exosomes are extracellular vesicles produced by all cells with a remarkable ability to efficiently transfer genetic material, including exogenously loaded siRNA, to cancer cells. Here, we report on a bioreactor-based, large-scale production of clinical-grade exosomes employing good manufacturing practice (GMP) standards. A standard operating procedure was established to generate engineered exosomes with the ability to target oncogenic Kras (iExosomes). The clinical-grade GMP iExosomes were tested in multiple in vitro and in vivo studies to confirm suppression of oncogenic Kras and an increase in the survival of several mouse models with pancreatic cancer. We perform studies to determine the shelf life, biodistribution, toxicology profile, and efficacy in combination with chemotherapy to inform future clinical testing of GMP iExosomes. Collectively, this report illustrates the process and feasibility of generating clinical-grade exosomes for various therapies of human diseases.

Authors

Mayela Mendt, Sushrut Kamerkar, Hikaru Sugimoto, Kathleen M. McAndrews, Chia-Chin Wu, Mihai Gagea, Sujuan Yang, Elena V. Rodriges Blanko, Qian Peng, Xiaoyan Ma, Joseph R. Marszalek, Anirban Maitra, Cassian Yee, Katayoun Rezvani, Elizabeth Shpall, Valerie S. LeBleu, Raghu Kalluri

×

Figure 4

Validation of GMP-grade iExosome efficacy in vitro.

Options: View larger image (or click on image) Download as PowerPoint
Validation of GMP-grade iExosome efficacy in vitro. (A) Representative d...
(A) Representative dot plot and (B) quantification of flow cytometry analysis of apoptosis in Panc-1 cells induced by MSC Ctrl Exo, MSC siScrbl iExo, MSC siKrasG12D–1 iExo, or BJ siKrasG12D–1 iExo after 48 hours, compared with untreated cells. Numbers represent the percentage of positive cells (n = 4 independent experiments, 1-way ANOVA compared with untreated). (C) KRASG12D transcript levels in Panc-1 cells treated with MSC Ctrl Exo, MSC siScrbl iExo, MSC siKrasG12D–1 iExo, or BJ G12D–1 iExo after 3 hours, compared with untreated cells (n = 4 independent experiments, 1-tailed unpaired t test). (D) Representative TEM of MSC exosomes, after electroporation, using either research buffer (RB) or clinical buffer (CB). Scale bar: 100 nm. (E) Representative dot plot of flow cytometry analyses and quantification of apoptosis in Panc-1 cells untreated or treated for 48 hours with MSC siKrasG12D–1 iExo electroporated using either RB or CB. Numbers represent the percentage of positive cells (n = 4 independent experiments, 1-way ANOVA compared with untreated). (F) KRASG12D transcript levels in Panc-1 cells (n = 3 independent experiments, 1-tailed unpaired t test). The mean ± SEM is depicted. *P < 0.05, **P < 0.01, ****P < 0.0001. See Supplemental Source Data 1 and 2.