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 cancerMinh Le, PhD Assistant Professor Department of Biomedical Sciences City University of Hong K

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

Andaloussi et al, Nature Reviews, 2013

• 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

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

Purification of RBCEVs

Usman et al, Nature Communications 2018

Waqas M Usman Tin Pham

Characteristics of RBCEVs

Protein contents

Usman et al

• Size distribution of EVs from 3 donors (grey: SEM) were determined using Nanosight NS300

• Image of EVs was captured using transmission electron microscopy (TEM), scale bar: 200 nm

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

• MOLM13 cells were also transfected with ASOs using Lipofectamin 3000 (Lipo) from Thermo Fisher Scientific

or InterferIn (Inte) from PolyPlustransfection

• Student’s t-test: n.s., non-significant;

*** P < 0.001 and **** P < 0.0001

10080604020

RBCEVs deliver ASOs to leukemia cells at low toxicity

Usman et al

• 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

Commercial reagents

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

Usman et al

• 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

• P values were determined using one-way ANOVA test

miR-125b inhibition upregulated BAK1 and suppressed proliferation of MOLM13 cells

Usman et al

• 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

Biodistribution of RBCEVs upon a systemic administration

Usman et al

• IVIS images of the organs 24 hours after 2 i.p injections (24 hours apart) of 3.3 x 1012 DiR-labeled RBCEVs or the supernatant from the last wash of labeled EVs

• N = 4 mice

Uptake of RBCEVs by bone marrow cells

Usman et al

• 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 2 i.p injections (24 hours apart) of 3.3 x 1012 VVT-labeled RBCEVs

• N = 4 mice

Delivery of 125b-ASO suppresses the leukemia progression

Delivery of 125b-ASO suppresses the leukemia progression

Usman et al

FACS analysis of GFP from leukemia cells in the bone marrow

Delivery of Cas9 mRNA and gRNAs to leukemia

cells for genome editing

Usman et al

Cas9 mRNAloaded 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.

Delivered Cas9 mRNA was translated into Cas9 protein

Usman et al

Delivery of Cas9 mRNA and gRNA to leukemia

cells for genome editing

Usman et al

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

• 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

Michael Yang Linfeng Huang Chun Kit Kwok Leo Chan Liang Zhang Zongli Zheng Likun Wei San Chan

Queen Elizabeth Hospital

& Hong Kong Red Cross

William Cho Hazel Kwok Victor Ma

Hong Kong University Anskar Leung

CityU Applied Research Grant Hong Kong Research Grant Council Early Career Development Scheme

Natural Science Foundation of China Grants

Whitehead Institute Harvey Lodish

Cornell University Andrew Grimson Kristy Richard Harvard Medical School Judy Lieberman

UC Berkeley Randy Schekman Morayma Temoche