CD19 Chimeric Antigen Receptor-Exosome Targets CD19 Positive B-lineage Acute Lymphocytic Leukemia and Induces Cytotoxicity
Abstract
:Simple Summary
Abstract
1. Introduction
2. Results
2.1. Transfection of CD19 CAR Plasmids into HEK293T Producer Cells
2.1.1. Characterization of CD19 CAR Plasmid
2.1.2. Transfection of Producer Cell Line
2.2. Confirmation of CD19 CAR Plasmid into the Transfected Producer Cell Line
2.3. Characterization of Purified Exosomes
2.4. Confirmation of CD19 CAR Molecules Expression in Harvested Exosomes
2.5. Delivery of Exo-CD19 CAR Molecules into the Target Cells
2.6. Exo-CD19 CAR Effect on CD19-Negative and CD19-Positive Cells
2.7. Exo-CD19 CAR Exposure Induces Pro-Apoptotic Genes in CD19-Positive B-Cell Leukemia
3. Discussion
4. Materials and Methods
4.1. Cell Lines
4.2. CD19 CAR Plasmid Isolation
4.2.1. Bacterial (Escherichia coli) Culture and Plasmid Isolation
4.2.2. CD19 CAR Plasmid Verification by Semi-Quantitative PCR
4.3. Production of CAR-Exosomes in HEK293T Parent Cells
4.3.1. Plasmid Transfection, Enrichment of Transfected Cells by FACS Cell Sorting, and Fluorescence Microscopy
4.3.2. Cell Staining to Analyze Transfection Efficiency by Flow Cytometry
4.4. Exosomes (Exo-WT and Exo-CD19 CAR) Production and Isolation
4.4.1. Exosomes Production
4.4.2. Preparation of Exo-Free FBS
4.4.3. Exosomes Isolation
4.4.4. Exosomal Markers CD63 and CD81 Expression Analysis by Flow Cytometry
4.4.5. Expression of CD19 CAR Markers Proteins on Exosomes by Flow Cytometry
4.4.6. Exosomal RNA Extraction
4.5. Exo-CD19 CAR Induced Cytotoxicity (Contact-Dependent) in CD19-Positive B-Leukemia Cells
4.5.1. Gene Expression by qPCR
4.5.2. Cytotoxicity Measurement by MTS Assay
4.6. Statistical Analysis
5. Conclusions
6. Patents
Supplementary Materials
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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SN | Gene Name | Gene ID | Primers |
---|---|---|---|
1 | eGFP | JQ064507.1 | For 5′-CTGGTCGAGCTGGACGGCGACG-3′ |
Rev 5′-CACGAACTCCAGCAGGACCATG-3′ | |||
2 | CD8a | NM_001768.6 | For 5′-CACGACGCCAGCGCCGCGACCACC-3′ |
Rev 5′-GGGTGATAACCAGTGACAGGAGAA-3′ | |||
3 | CD28 | AJ937363.1 | For 5′-GGAGGGGGGACCAAGCTGGAGA-3′ |
Rev 5′-TGCAGACTGTTCATTTTTAAG-3′ | |||
4 | β-actin | NM_001101.4 | For 5′-GTCCTCTCCCAAGTCCACACA-3′ |
Rev 5′-CTGGTCTCAAGTCAGTGTACAGGTAA-3′ |
SN | Gene Name | Gene ID | Primers | UPL # |
---|---|---|---|---|
1 | BAD | AF031523.1 | For: 5′-CGAGTTTGTGGACTCCTTTAAGA-3′ | 78 |
Rev: 5′-CACCAGGACTGGAAGACTCG-3′ | ||||
2 | BAX | U19599.1 | For: 5′-CAAGACCAGGGTGGTTGG-3′ | 55 |
Rev: 5′-CACTCCCGCCACAAAGAT-3′ | ||||
2 | Caspase-3 | DD346274.1 | For: 5′-AATGGACCAGGACGATGAAG-3′ | 35 |
Rev: 5′-CATCTCATCACCCACTGCTC-3′ | ||||
4 | GAPDH | NM_002046.3 | For: 5′-AGCCACATCGCTCAGACAC-3′ | 60 |
Rev: 5′-GCCCAATACGACCAAATCC-3′ | ||||
5 | IFN-g | X13274.1 | For: 5′-GGCATTTTGAAGAATTGGAAAG-3′ | 21 |
Rev: 5′-TTTGGATGCTCTGGTCATCTT-3′ | ||||
6 | TNF-a | X02910.1 | For: 5′-GTCCAGGCTTGTCCTGCTAC-3′ | 7 |
Rev: 5′-AGTCCTGAGGCCTGTGTTTG-3′ | ||||
7 | IL-2 | S77834.1 | For: 5′-AAGTTTTACATGCCCAAGAAGG-3′ | 65 |
Rev:5′-AAGTGAAAGTTTTTGCTTTGAGCTA-3′ |
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Haque, S.; Vaiselbuh, S.R. CD19 Chimeric Antigen Receptor-Exosome Targets CD19 Positive B-lineage Acute Lymphocytic Leukemia and Induces Cytotoxicity. Cancers 2021, 13, 1401. https://doi.org/10.3390/cancers13061401
Haque S, Vaiselbuh SR. CD19 Chimeric Antigen Receptor-Exosome Targets CD19 Positive B-lineage Acute Lymphocytic Leukemia and Induces Cytotoxicity. Cancers. 2021; 13(6):1401. https://doi.org/10.3390/cancers13061401
Chicago/Turabian StyleHaque, Shabirul, and Sarah R. Vaiselbuh. 2021. "CD19 Chimeric Antigen Receptor-Exosome Targets CD19 Positive B-lineage Acute Lymphocytic Leukemia and Induces Cytotoxicity" Cancers 13, no. 6: 1401. https://doi.org/10.3390/cancers13061401