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
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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

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Figure 6

Efficacy of large-scale produced-GMP iExosomes in combination with gemcitabine.

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Efficacy of large-scale produced-GMP iExosomes in combination with gemci...
(A) Representative dot plot and (B) quantification of flow cytometry analyses of apoptosis in Panc-1 cells induced by MSCs siKrasG12D–2 iExo, comparing low scale (LS) or high scale (HS) electroporation of MSC exosomes. Numbers represent the percentage of positive cells (n = 3 independent experiments, 1-way ANOVA compared with untreated). (C) KRASG12D transcript levels in Panc-1 cells incubated 3 hours with MSCs siKrasG12D–2, comparing LS or HS electroporation of MSC exosomes (n = 3 independent experiments, 1-tailed unpaired t test). (D) qPCR of siRNA for KrasG12D (same siRNA sequence from 2 purchasing sources, siKrasG12D–1 and siKrasG12D–2 for source 1 and 2, respectively) in the indicated samples (n = 3 distinct samples treated on the same day; input siRNA: n = 1). The data are presented as 1/Ct and mean ± SD. CB, clinical buffer; RB, research buffer; T, Triton X-100; RN, RNase A. (E) Kaplan-Meier curve indicating the survival of KPC689 mice after tumor induction in the listed treatment groups (CB/PBS [n = 7], Control Exo [n = 7], gemcitabine [n = 8], MSC siKrasG12D–2 iExo [n = 8], gemcitabine + MSCs siKrasG12D–2 iExo [n = 8]; log-rank [Mantel-Cox] test). (F) Kaplan-Meier curve indicating the survival of KPC689 mice after tumor induction in the listed treatment groups (n = 7 mice in each of the listed groups; log-rank [Mantel-Cox] test). Unless otherwise specified, mean ± SEM is depicted. Unless stated otherwise, 1-way ANOVA, comparing experimental groups to control groups, was used to determine statistical significance. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. See Supplemental Source Data 1 and 2.