The Problem: Biomedical Monitoring of Gene Dynamics
How is mammalian gene expression analyzed?
There is an increasing demand in biomedical research for techniques to monitor the dynamics of multiple genes over several time points. However, current methods such as gene reporters are limited to a few genes of interest or require sample destruction in the case of transcriptomic analysis.
In basic research, there is an overwhelming diversity of whole-cell therapeutic approaches waiting to enter clinical pipelines:
CAR-T-Cells
Engineered Macrophages
Engineered B-Cells
Beta-Cell Transplants
Neuronal Stem-Cell Therapy
Our aim was to consolidate the various biomarkers to one universal exportable biomarker, enabling minimally-invasive longterm monitoring of any cell type.
Currently, especially after transplantation, monitoring capabilities of current techniques are minimal. Cells can be isolated and lysed for further analysis of the cell state. This technique holds many problems as it is invasive, prevents long-term monitoring and consumes the cells, therefore destroying the therapeutic agents in cell therapy.
Our Solution:
Analysis of Living cells by Vesicular Export
ALiVE is a diagnostics platform for the Analysis of Living cells via Vesicular Export. More specifically, ALiVE allows monitoring of mammalian cells by exporting RNA gene markers from the cell of interest in engineered protective vessels. These can be purified from surrounding fluids and enable live monitoring of gene expression by analyzing the vesicles' content.
Scientific Design Criteria
Work distribution
We were able to approach the lab work from multiple sides simultaneously. The modules were designed to work independently of each other, allowing us to proceed with multiple ideas without being hindered by setbacks in other parts of the project. Beforehand, together with professors and PhD-students, we designed specific assays for each workpackage, making troubleshooting straight forward.
The final platform consists of five main modules:
Information Encoding
Cargo Loading
Vesicle Secretion
Vesicle Isolation
Cargo Analysis
We developed multiple options for every module for the final user to choose from depending on the intended application.
Information Encoding
Our goal is to export a molecule that gives insight into the current cell state.
Proteins
The status of a cell strongly depends on the proteome. Therefore the most straightforward answer would be to export proteins with an export sequence.
Protein characterization is complex, hard to multiplex, and cannot be amplified. Additionally, some functions are not performed by proteins.
RNA
Another molecule with similar "encoding potential" that we looked into was mRNA. Its presence gives direct information about the expressed genes, and it allows a high throughput analysis via next-generation sequencing techniques.
RNA as exported molecule allows high-throughput sequencing.
The choice of RNA as a molecule of interest gives multiple options depending on the application. We could either export mRNA directly out of the cytosol to enable omics-like workflows or mark specific genes with short RNA-markers. As RNA is made up of 4 bases, the encoding potential of an RNA strand of length n is 4n. This implies an enormous advantage over gene expression tracking with combinatorial fluorescent protein labeling methods currently used where only a few dozen different signals can be differentiated (Cook et al. 2019; Pontes-Quero et al. 2017).
Cargo Loading
We employ 2 orthogonal RNA-binding proteins, L7Ae and MCP, as RNA-adaptors.
In the spirit of modularity, we designed independent vesicular loading mechanisms.
The first mechanism is essentially an RNA-binding-protein fused to a vesicle marker. The RNA-binding-protein and therefore the RNA molecule itself will be loaded directly into the vesicles.
The second mechanism is designed to be more modular. It involves a set of coiled-coil domains which attach to its counterpart via electrostatic interactions, non-covalently. This interaction is used to direct RNA-binding-proteins to the cellular membrane for export, making the use of multiple RNA-binding-proteins much simpler.
The third mechanism is designed to be non-invasive. By not actively exporting "active" mRNA from the cell, it is minimally invasive and opens up the possibility for seamless analysis of living cells.
Vesicle Secretion
The transport of RNA within bodily fluids is a significant part of ALiVE. To protect RNA, we had to enable the cell to package and secrete the RNA in protective cargo vesicles. Initially, we chose to hijack the endogenous vesicular export pathway. To further pursue our goal of a modular platform, we decided to work on multiple vesicle systems in parallel. Another method involves synthetic vesicles made up of structural gag-proteins from the HI-Virus making the secretion bioorthogonal. These vesicles are stripped of infectious proteins and safe under european safety regulations (S1).
We tested the different vesicles regarding particle formation and cargo loading efficiency, purification capabilities, cell toxicity, and bioorthogonality.
Vesicle Isolation
Bodily fluids in organisms contain many vesicle-sized objects. To use ALiVE for efficient cell monitoring, one would need the information-loaded vesicles to be distinguishable from naturally occurring objects of the same size. To achieve this goal, we designed different purification constructs for each vesicle, enabling their isolation and purification out of varying fluid volumes of cells by following simple step-by-step protocols.
Cargo Analysis
As we envision ALiVE to be a platform that covers a broad field of applications, we had to design our analysis methods accordingly. We divided our analytic methods into two main application fields. For gene-specific markers, we developed methods based on the fact that the sequence of the possible RNA molecules inside the vesicles are known beforehand. The direct export of mRNA out of the cytosol meant that the number of possible sequences was too vast to use the same methods as for specific gene markers. Therefore, we planned to used next-generation sequencing techniques to analyze the entirety of our exported information.
Beta- and T-cell lines
To finalize our project, we aimed to apply our proof of principle to more application-oriented cell lines. Here were decided to test two cell lines:
Beta-Cells
As we envision an application in beta-cell transplantants, we planned to apply ALiVE to a mouse beta cell line.
T-Cells
While designing ALiVE, CAR-T cells immediately struck our interest, as our system could be implemented during their genetic modification. We therefore planned to apply ALiVE to a human T-cell line.
