Project Lethe and the innovative gene therapy approach it proposes against Alzheimer’s Disease is a project and idea worthy of further research and investigation. Firstly, it is essential to begin with research to solidify the factual ground Project Lethe stands on, creating an unshakeable scientific foundation. In the beginning of our journey, we tested and evaluated some of the key parts of our project. Building upon our initial steps, our team has carefully considered and planned the groundwork experimental procedures required for this idea to become a reality. Following this, we plan to lay down our suggestion for the transformation of the project into an industrially produced pharmaceutical, the standardization of the production line, and how that final form would look.
We begin with the rAAV triple transfection process and rAAV viral particles production. For this process, optimization measures we plan on taking concern the efficiency of the components used. Specifically, we aim to use a cell line specialized for rAAV production, for example, the AAV293 cell line. Simultaneously, we plan on incorporating an equally specified transfection reagent like FectoVIR®-AAV. On a second note, we found browsing through many sources that for HEK293T derived cell lines to be cultured as adhesive cell lines is very difficult due to their easy detachment from the dish. The pre-coating of cell culture plates with the cationic polymer poly-L/D-lysine is recommended to prevent cell loss.
The isolation of exosomes using Exo-Prep® or other similar reagents remains unchanged. However, a step for the characterization and quantification of exosomal yield must be added. Quantification can be performed through Western Blotting or Flow Cytometry using standard surface proteins of exosomes as markers. The use of a corresponding kit like Exo-TEST® based on ELISA technique could aid us in the characterization. Transmission Electron Microscopy (TEM) is also helpful for this purpose.
Equally important is the quantification of the rAAV2 viral particles yield through a full to empty ratio test. After a complete purification and isolation process of the viral particles from the exosomes (using a specified technique, e.g., iodixanol gradient), it is critical that a quantification of the capsid proteins (through ELISA technique) and quantification of viral DNA (through qPCR) is performed. This way, a ratio can be inferred regarding the number of capsids carrying viral DNA and the number of capsids that do not. This offers us an image of the actual AAV titer. The processes described in this paragraph all aid in the accurate calculation of the appropriate dosage of vexosomes during the treatment testing.
The next step is the treatment. The cells that will originally receive the treatment must simulate the model of Alzheimer’s Disease as closely as possible. A widely used option is the usage of HEK293T cells modified to express Tau protein (e.g., HEK293/Human Tau-K18 (GFP) Stable Cell Line), which then can be hyperphosphorylated by a biological factor; Calyculin A has proved a sufficient strategy already. This model provides the opportunity to track the effects of microRNA-195 on the levels of PME-1, PP2A levels, methylation and activity, and eventually Tau phosphorylation and aggregation. On the same note, BACE-1 and APP levels should also be examined in relation to hsa-mir-195-5p's action and its anti-Αβ-amyloid formation properties. For a broader study of the effects of this microRNA overexpression, a neural cell line exhibiting AD pathology can be used (e.g., Alzheimer’s In A Dish™: 3D Neural Stem Cell Models of Alzheimer's Disease). The effects of mir-195-5p on the proteins’ level and state can be measured through FACS analysis using a respective conjugated antibody for each use described above. Ideally, for PME-1, BACE-1, and APP, qPCR for their respective mRNA levels is advisable, considering mir-195-5p targets them directly. Of course, the overexpression of hsa-mir-195-5p will be assessed using qPCR. It is crucial that a dosage-response curve is carefully calculated based on the dosage and the measured effect in the cells. This way, we will be able to pinpoint the amount of vexosomes that is required for a capable dose.
In vivo trials in an animal model are the next step. Here, a more accurate and valuable dosage-response ratio can be inferred. A living organism also provides the ideal ground to test the vexosome neuron-targeting abilities when it is coated with the RVG29 peptide. Surface modification of vexosomes will be achieved through click-chemistry and a subsequent step for purification using HPLC column chromatography. The modified vexosomes will be injected into the bloodstream, and the spread of them in the body, especially in regions of the brain, can be studied using fluorescence, perhaps using fluorescent labeling (e.g., ExoSparkler Exosome Membrane Labeling Kit-Green). In animal models, our alternative method for administration—intranasal injection—will be researched. Intranasal administration is a promising, patient-friendly approach for brain-targeting drugs and is compatible with exosome-based therapies. This approach might render the present neuro-targeting properties of our vexosomes less useful or require different targeting modifications to navigate the vexosomes to certain parts of the brain. The required dosage might also be different, so it should be extensively investigated. Other studies show a potential negative effect of uncontrolled overexpression of miR-195 on neurite outgrowth and synaptic plasticity. Establishing mechanisms to tightly control miR-195 expression levels is vital, preventing overexpression that could lead to potential synaptic dysfunction. This may involve inducible systems that allow for temporal control. Extensive testing in animal models of Alzheimer’s disease to assess the long-term safety and efficacy of controlled miR-195 overexpression. The most significant opportunity animal testing will provide, though, is an assessment of the adverse effects of this therapy on the CNS and other tissues of the body alike. This stage will be one of the most important, as it involves extensive monitoring of the animals’ responses to the drug. Depending on the results, our drug will be modified accordingly to erase any possible side effects.
Clinical trials are the last step. Carefully designed clinical trials with patient-specific considerations, evaluating miR-195 therapy’s impact on cognitive function, neurodegeneration, and biomarkers of Alzheimer's pathology. Only if our drug is deemed suitable for human consumption will our project become a reality, helping people suffering from AD worldwide.
For a viable industrial level of production, some of the production steps of modified vexosomes must be altered. Most notably, the AAV production and vexosome isolation must be done with iPSC cell lines cultured in large numbers in bioreactors with optimal conditions, which will be thoroughly investigated. Again, this product will be different from the one described previously, so the experimental process must be repeated. The choice for iPSCs is made for their high availability, ease of growing in vitro, and transfection capabilities.
We always keep in mind that the scientific value of our results will reside in the strong repeatability of our results. Reaching that stage will require caution at every step, using control samples whenever it is possible for easy backtracking of any possible problems and their easy resolution.