How are mammalian cells monitored in basic research and diagnostics?
Gene Reporters
Fluorescent gene reporters visualize the gene expression of genes of interest.
Limitations
Multiple time points but limited multiplexing
-Omics Methods
-Omics methods such as transcriptomics reveal a cell´s transcriptomic state
Limitations
Multiplexing but destructive end-point measurement
Invasive
State of the Art Methods are either destructive or limited in their multiplexing capabilities
Our Solution:
Analysis of Living cells by Vesicular Export
We are developing a diagnostic tool for minimally-invasive monitoring of transplanted cells over time by using vesicles to export transcriptomic information.
How does it work?
Genetically Encoded Reporters
RNA gene reporters contain a unique RNA sequence (gene-identifier) and a structural motif (export-handle). They can be inserted into the 5’ UTR of any gene of interest.
Gene Reporter Maturation
The RNA gene reporter is present in the cytosol where it folds into a stem-loop structure. It is now ready to be exported out of the cell.
Vesicle loading
RNA binding proteins bind stem-loop structures highly specific. By using a set of diverse adapters, the RNA-binding proteins can be exported out of the cell, thereby secreting the RNA reporters. Here, the endogenous exosomal pathway can be hijacked, or fully orthogonal gag-vesicles can be used.
Vesicle Formation
The vesicles are formed either directly at the cell membrane as in the case of VLPs, or, as for exosomes, at the endosomal membrane.
Vesicle Secretion
The vesicles are continuously secreted over time. Both lipid-based containers protect RNA gene reporters from conditions outside of the cell. After vesicle isolation and lysis, the RNA can be analyzed.
Evolution of Medical Therapies Requires Novel Diagnostic Tools
Cell therapies are revolutionizing medicine. However, new treatments call for novel diagnostic techniques. Transplanting living therapeutic agents requires accurate monitoring over long periods. We envision ALiVE to enable long term live-cell monitoring non invasively.
There is currently a lot of research going into different fields of cell therapy and cell replacement therapy. Some of the planned cell therapies are still in a very early stage, while others are already close to or already in clinical trials
Neural Stem Cell Therapy
treating diseases like Parkinson
Very early stage
CAR-T - cells
During akute lymphoblastic leukemia lymphoblasts grow uncontrollably without causing an immune response. CAR-T - cell therapy aims to modify T - cells to detect and destroy the lymphoma in the patient after transplantation.
Modification of patient T - cells with Chimeric Antigen Receptor (CAR)
Reinsertion of modified T - cells to kill cancer cells
Clinical Trials in the US
Beta Cells:
Insulin secreting pancreatic cells get destroyed during Type 1 Diabetes. Transplantation of functioning beta cells could eliminate the necessity of insulin injection therapy.
Treatment for Type 1 Diabetes
Transplantation of insulin - producing beta cells to accomodate for lack of insulin production
Clinical Trials
Vision: ALiVE as a diagnostic tool for beta cell therapy
Type I Diabetes patients have to administer insulin multiple times a day, for the rest of their life. Cell therapy researchers are thus working on beta-cell transplants to replace the insulin injections. To this end, we evaluated ALiVE in cooperation with experts and companies in the field.
We imagine the application of ALiVE in 2 ways:
- To monitor the differentiation process of a therapeutic cell
- As a diagnostic tool for non-invasive monitoring of cell transplants
Our goal is for ALiVE to assist in the development and practice of cell therapies, as in the example of beta-cell therapy, as a diagnostic tool.
Entrepreneurship
We analyzed the potential of ALiVE as diagnostic tool for cell therapies, specifically for the case of CAR-T cells. To this end, we evaluated ALiVE according to different critera in cooperation with experts and companies in the field.
During the process of planing our vesicle isolation methods we contacted Prof. Dr. Hagn, a membrane protein specialist. Integrating his feedback, we designed a modified exosomal membrane protein for vastly increased isolation.
The membrane protein designed by integrating Prof. Dr. Hagns feedback gave us the opportunity to isolate exosomes with great success. This biobrick, a part of our modular part collection, is now open to use for any iGEM team, making vesicle analysis alot more accessible.
Our part collection comprises loading and export mechanisms for two vesicle secretion pathways. Additionally the toolset we designed allows the usage of multiple RNA oading proteins that can be loaded into the vesicles over a modular adapter system.
By breaking down and presenting our project in an understandable manner at a science communication event and organising a lecture and experiments for school students, we made science accessible to the public while tremendously improving our science communication skills.
