Our goal for ALiVE is to create a diagnostic tool in the field of cell transplant medicine and basic medical research in general to monitor cells minimally-invasive. Therefore, as a first demonstration, we proved its functionality in a mammalian human embryonic kidney cell line called HEK 293T.
To ensure the applicability of ALiVE, we analyzed the following criteria of our system in HEK293T cells to develop a proof of principle:
Secretion
The first aspect we had to test was the formation and secretion of the Gag-based VLPs and CD63-containing exosomes. For this, we designed analytic assays based on a split luciferase assay that allowed us to detect correctly formed vesicles and calculate export rates with our in silico model (read more). This "HiBiT-Luciferase-Assay" is shown for VLPs and exosomes in figure 1 The data shows that both vesicles with loaded RNA binding protein L7Ae are successfully secreted in HEK293T cells. The exosomal secretion pathway led to a low export rate, while the VLPs were exported in high amounts.
Purification
For further analysis of the secreted vesicles and their content, we designed vesicle specific purification methods. For exosomes, we developed a novel purification construct with the help of a membrane protein expert (read more). This allows Nickel-NTA purification of exosomes for the first time. For VLPs, we established a heparin-based purification method. The purification of both vesicles was analyzed over the previously introduced HiBit assay. The successful purification für His-tagged exosomes is shown in figure 2 .
Time-resolution
The most crucial aspect of our project was the cargo loading into the vesicles. To analyze the vesicles' content, we established a harvesting method gentle to the cells, allowing us to take multiple measurements from the same wells. After extracting and reverse transcribing the cargo RNA from the vesicles, we analyzed and quantified the amount of exported cargo transcript using qPCR, thus achieving the primary goal of our project.
We harvested supernatant from the same cells at three different time points over 72h and were able to show constant mRNA levels in our vesicles, proving ALiVE's functionality.
ALiVE allows minimally-invasive longitudinal cell monitoring
Viability
Following the initial proof of concept, we wanted to investigate safety concerns. The first concern was ALiVEs impact on cell viability. Herefore, we performed an LDH-based viability assay over multiple timepoints evaluating ALiVEs longterm impact on vesicle secreting cells. The data in figure 4 shows minimal differences between cells that were transfected with ALiVE versus control groups.
ALiVE is not harmful to the affected cells
Collateral transfection
As we envision ALiVE to be a diagnostic tool for transplant medicine, its impact in an organism-type environment had to be analyzed. More precisely, due to the vesicles being present in bodily fluids for extended amounts of time, their collateral transfection effect had to be tested. Collateral transfection rate describes the quantity of RNA exported that gets taken up by other surrounding cells. To check this, we designed a multi-timepoint reuptake assay, based on our exported transcript encoding for a luciferase. Due to this fact, we could monitor the collateral transfection in two ways. First, we performed qPCR from the cells taking up vesicles to directly determine the amount of uptaken RNA. Second, we tested the expression of the encoded luciferase in previously untransfected cells after treating them with our vesicles. The data from qPCR in figure 5 shows minimal reuptake of our vesicles, with the VLPs being slightly more infectious.
