Harnessing extracellular vesicles from human red blood cells for gene therapies gene therapies cancer

Harnessing extracellular vesicles from human red blood cells for gene therapies against cancer Minh Le, PhD Assistant Professor Department of Biomedical Sciences City University of Hong Kong Extracellular vesicles mediate intercellular communication • Extracellular vesicles (EVs) are membrane vesicles secreted by many cell types • EVs include exosomes derived from the multivesicular bodies and microvesicles derived from the cellular membranes • EVs deliver proteins and RNAs for intercellular signaling Andaloussi et al, Nature Reviews, 2013 Extracellular vesicles based gene therapies • EVs are natural carriers of RNAs • EVs have been used to deliver siRNAs, miRNAs, mRNAs and plasmids • EVs offer great biocompatibility: • Robust uptake by many cell types • Low toxicity • Low immunogenicity • EVs may avoid phagocytosis and multidrug resistance Common sources of EVs for therapies • Human cell lines and stem cells: expandable in culture • Oncogenic/transformation risks • Huge expenses on cytokines and other supplements • Human dendritic cells and fibroblasts: safe, allogenic  how to get enough cells? • Bovine milk: low cost, unlimited • Interspecies transfer of proteins and RNAs  immunogenic? • Promoting metastasis? (Mathivananan et al) • Human plasma: readily available, allogenic • Variable and unpredictable components • Allogenic plasma may contain disease-promoting EVs Extracellular vesicles from red blood cells • RBCs are the most abundant cell type (84% of all cells) in the body • RBCs can be obtained readily from any human subject, and have been used safely and routinely in the hospital for blood transfusions over decades • RBCs lack both nuclear and mitochondrial DNA  no risk of gene transfer • Treatment with calcium ionophore induces massive EV release by RBCs Purification of RBCEVs Usman et al, Nature Communications 2018 Waqas M Usman Tin Pham Characteristics of RBCEVs Morphology Size distribution • • Protein contents Size distribution of EVs from donors (grey: SEM) were determined using Nanosight NS300 Image of EVs was captured using transmission electron microscopy (TEM), scale bar: 200 nm Usman et al Uptake of RBCEVs by leukemia MOLM13 cells Uptake of PKH26-labeled EVs by MOLM13 cells • • • • • MOLM13 cells are acute myeloid leukemia cells GFP is overexpressed in MOLM13 cells using lentivirus PKH26 is a fluorescent membrane dye binding to EVs MOLM13 cells were incubated with PKH26-labeled EVs for 24 hours Images were captured using confocal microscopy Scale bar, 20 µm Usman et al RBCEVs deliver ASOs to leukemia cells at high efficiency • • FAM+ cells % • 100 80 60 40 20 • FAM-ASOs: scrambled ASOs labeled with FAM fluorescence Uptake of FAM-ASO-loaded EVs by MOLM13 cells were analyzed using FACS MOLM13 cells were also transfected with ASOs using Lipofectamin 3000 (Lipo) from Thermo Fisher Scientific or InterferIn (Inte) from PolyPlus transfection Student’s t-test: n.s., non-significant; *** P < 0.001 and **** P < 0.0001 Usman et al RBCEVs deliver ASOs to leukemia cells at low toxicity Commercial reagents • • Cell death was quantified by FACS analysis of Propidium iodide (PI) staining Student’s t-test: n.s., non-significant; ** P < 0.01 and **** P < 0.0001 Usman et al miR-125b is a common oncogene in cancer • miR-125b targets the tumor-suppressor p53 and about 20 genes associated with p53 to regulate the activity of the p53 pathway (Le et al, G & D 2009, Le et al PLoS Gen 2011) • miR-125b is essential for survival of normal cells and many types of cancer cells (Le et al, G & D 2009, Yin et al, Exp Cell Re 2015) • miR-125b acts as an oncogene in leukemia, lymphoma, prostate cancer, lung cancer, breast cancer, glioma, kidney cancer, gastric cancer and retinoblastoma (Yin et al, Exp Cell Re 2015) RBCEVs deliver ASOs to leukemia MOLM13 cells for miR-125b inhibition miR-125b • • • • miR-125a 125b-ASO: antisense oligonucleotides complementary to miR-125b and partially to miR-125a UE-EVs: unelectroporated RBCEVs NC-ASO E-EVs: RBCEVs electroporated with negative control ASOs Usman et al P values were determined using one-way ANOVA test miR-125b inhibition upregulated BAK1 and suppressed proliferation of MOLM13 cells BAK1 upregulation • • Proliferation suppression BAK1 is a validated target of miR-125b that induces apoptosis P values were determined using one-way ANOVA test (left) or student’s t-test (right): *P < 0.05; **P < 0.01 Usman et al Biodistribution of RBCEVs upon a systemic administration • • IVIS images of the organs 24 hours after i.p injections (24 hours apart) of 3.3 x 1012 DiRlabeled RBCEVs or the supernatant from the last wash of labeled EVs N = mice Usman et al Uptake of RBCEVs by bone marrow cells • • • EVs were labeled with vivotrack-680 dye (VVT) FACS analysis of VVT fluorescence (APC -Cy5.5) in bone marrow cells from the mice 24 hours after i.p injections (24 hours apart) of 3.3 x 1012 VVT-labeled RBCEVs N = mice Usman et al Delivery of 125b-ASO suppresses the leukemia progression IVIS analysis of bioluminescence from leukemia cells in live mice • – days after the treatment with 3.3 x 1012 RBCEVs containing NC-ASO (N = mice) or 125bASO (N = mice) every days relative to the signals before the treatment started (day 0) Usman et al Delivery of 125b-ASO suppresses the leukemia progression FACS analysis of GFP from leukemia cells in the bone marrow Usman et al Delivery of Cas9 mRNA and gRNAs to leukemia cells for genome editing Cas9 mRNA in EVs Cas9 mRNA in MOLM13 cells Cas9 mRNA loaded EVs Cas9 mRNA levels in RBCEVs treated with RNase (left) or in MOLM13 cells treated with unelectroporated EVs (UE-EVs) or EVs loaded with 3-6 pmol Cas9 mRNA (right) ***P < 0.001: Student’s t-test Usman et al Delivered Cas9 mRNA was translated into Cas9 protein Usman et al Delivery of Cas9 mRNA and gRNA to leukemia cells for genome editing miR-125b BAK1 MOLM13 cells treated with unelectroporated EVs (UE-EVs) or EVs loaded with Cas9 mRNA and mir-125b-targeting gRNA for 48 hours *P < 0.05; ****P < 0.0001: Student’s t-test Usman et al Summary • RBCEVs is a versatile delivery system for therapeutic RNAs, including both short RNAs and mRNAs • Anti-miR-125b ASO delivered by RBC EVs inhibits miR-125b function and suppresses cell growth in vitro and leukemia progression in vivo • RBCEVs also deliver Cas9 mRNA and gRNA for genome editing • ASOs and CRISPR-Cas9 can be designed and programmed to target any gene of interest, including undruggable targets • RBCEV-delivery system is suitable for clinical applications because: • RBCs are readily available from blood banks and patient’s own blood • Large amount of RBCEVs (1013-1014) can be obtained from each blood unit • RBCEVs are safe as the enucleated RBCs are homogeneously devoid of DNA • RBCEVs are nontoxic and likely non-immunogenic • RBCEVs are stable after multiple free-thaw cycles • Further development of RBCEVs coated with cancer-targeting peptides or antibodies could potentially deliver therapeutic RNAs to cancer cells specifically Summary Acknowledgement Le Lab CityU collaborators Queen Elizabeth Hospital Whitehead Institute Waqas Usman Jiahai Shi & Hong Kong Red Cross Harvey Lodish Tin Pham Michael Yang William Cho Louis Vu Linfeng Huang Hazel Kwok Boya Peng Chun Kit Kwok Victor Ma Abdullah Faqueer Leo Chan Hong Kong University Xin Zhang Liang Zhang Anskar Leung Thach Pham Zongli Zheng Alex He Chriss Chin Likun Wei A*STAR Singapore Kei Kim San Chan Ng Shyh Chang Cornell University Andrew Grimson Kristy Richard Harvard Medical School Judy Lieberman UC Berkeley Randy Schekman Morayma Temoche Milly Kim Vietnam Institute of Hematology Chinh Duong Funding Hong Kong Health and Medical Research Fund CityU Start-up and VPRT Grants CityU Applied Research Grant Hong Kong Research Grant Council Early Career Development Scheme Natural Science Foundation of China Grants ... Variable and unpredictable components • Allogenic plasma may contain disease-promoting EVs Extracellular vesicles from red blood cells • RBCs are the most abundant cell type (84% of all cells) in... sources of EVs for therapies • Human cell lines and stem cells: expandable in culture • Oncogenic/transformation risks • Huge expenses on cytokines and other supplements • Human dendritic cells and... and microvesicles derived from the cellular membranes • EVs deliver proteins and RNAs for intercellular signaling Andaloussi et al, Nature Reviews, 2013 Extracellular vesicles based gene therapies