mRNA Therapy
Abstract
After we successfully developed our Entry Vectors pASTERISK-HBA1 and pASTERISK-HBB, we are able to perform in vitro transcriptions for synthesis of our therapeutic mRNA. By comparing different variants of UTRs, we are able to design an optimized version of the DART VADAR technology. In the case of our intraoperative implementation, we postulated that pASTERISK-HBA1 generates mRNA molecules that are faster acting as pASTERISK-HBB.
☑ successfully perform in vitro transcription workflow for pASTERSIK-HBA1 and ASTERISK-HBB variants
☑ transfected cell lines and primary tumor tissue with our therapeutic mRNA
☑ comparison between variants at different time points shows differences between variants over the time
☑ evaluation of different transfection reagent enables cytotoxic effect
☑ ASTERISK-HBA1 leads to fast, but unstable expression over time
☑ ASTERISK-HBB leads to slow, but stable expression over time
In vitro transcription of therapeutic mRNA sensing and kill constructs
By implementing our optimized workflow for in vitro transcription using appropriate kits, we successfully performed RNA synthesis of our therapeutic mRNA constructs. By enzymatic capping with the ARCA Cap-Analogon and the Poly-A-Tailing via Poly-A-Polymerase, we aim to stabilize the mRNA to increase the therapeutic effect. After transfection of HEK295 cell lines, we identify an antiproportional behavior of expression.
Preparation of HEK293 cell culture
In order to test our therapeutic mRNA in vivo, we started cultivated cultivation of HEK294 cells by thawing and passaging. Our cultures were tested negative for different Mycoplasma contaminations at three different time points.
☑ no Mycoplasma spec. were detected in our cultures during the project
Preparation of tumor samples from primary tissue
According to our safety policy, we collaborated with the local hospital “Klinikum Bielefeld Mitte”. Dr. Matthias Simon provided fresh tumor samples after surgical removal from patients with unknown clinical background. Applying PCR, we excluded any contamination and thereby possible risks of the sample under strict safety and security measurements.
☑ no HIV, HCV and HPV genotypes or viral contamination were detected within the sample
As previously mentioned before in the Cancer Genome Sequencing section, we do perform Nanopore Sequencing experiments and transfection of primary tumor tissue as well. We aim to analyze the function of our therapeutic mRNA within patient samples and different limitations due to practical relevance for e.g. penetration depth and transfection efficiency. By comparing different transfection reagents and different variants at different time points after transfection, we aim to analyze the efficiency of our therapeutic approach via fluorescence microscopy and Flow cytometric analysis.
Fluorescence microscopy
☑ the fluorescence signals after ASTERISK-HBA1 transfection increase faster than the fluorescence signals after ASTERISK-HBB transfection at the same time ☑ in comparison to the Plasmid-based proof-of-concept we assume significantly faster expression after mRNA transfection ☑ based on the positive control, the transfection experiments were performed successfully ☑ transfection with different concentrations of transfection reagent lipofectamine does not significantly change fluorescence expression rate ☑ FACS results corresponds to the trend in microscopic images: HBB more stable than HBA ☑ cytotoxicity of mRNA construct does not change between same condition
FACS DIAGRAMS
The red signal represents the untreatened negativ control of HEK cell. The yellow signal represents the pASTERISK_HBA generated mRNA molecules. The blue signal represents the HBA variant in different Lipofectamin concentration. The light green seignal represents die HBB Variant with lower Lipofectamin concentration in comparison to the dark green singal, which represent the HBB Variant transfection with lipofectamin. All detections were performed 72 h after transfection.
RESULT - Because of the different Area and Signal intentisities, the HBB Variant expression seams to be more consistant. The HBA Varient seams to be less stable. We conclude, that the FACS results represents same results aus the fluoresecence analyzes. We proposed, that the ASTERISK_HBA Variant is more suitable for our intraoperative approach of direct mRNA therapy application.
☑ the fluorescence signals after ASTERISK-HBA1 transfection increase faster than the fluorescence signals after ASTERISK-HBB transfection at the same time
☑ in comparison to the Plasmid-based proof-of-concept we assume significantly faster expression after mRNA transfection
☑ based on the positive control, the transfection experiments were performed successfully
☑ transfection with different concentrations of transfection reagent lipofectamine does not significantly change fluorescence expression rate
☑ FACS is in alignment with the mikroscopic recordings: HBB is more stable HBA
☑ cytotoxicity of mRNA construct does not change between same condition
Final conclusion
mRNA molecules derived from pASTERSIK-HBA1 can facilitate acould lead to direct therapeutic effects. In case of intraoperative application, the sensing and killing switch is fast acting and provides direct targeting of cancer cells. Because of the low stability, the cytotoxic effect is time limited. In comparison, the transfection of mRNA molecules derived from the pASTERISK_HBB variant, leads to stable expression over time. The stability could be increased due to the postulated secondary structure. The decreased mRNA decay leads to stable expression of payloads. This observation leads us to prosume, that pASTERISK_HBB variants are better for long expression experiments.