Use of iPSCs in Alzheimer’s Disease Brain Inflammation
Prototype
CBI is caused by the brain heavily favoring an active immune response. Suppressive immune cells like regulatory t-cells (tregs) are decreased significantly in number, while active immune cells become harmful.
By artificially increasing the treg population, immune balance can be restored, CBI can be reduced, and AD progression can be significantly slowed.
This can be achieved by coding synthetic signaling pathways within the tregs genome. These pathways will activate the IL-2 gene when inflammation is detected. By activating this gene, tregs can produce their own IL-2 (a growth factor that increases treg population. )
1. Adult red blood cells are modified into induced pluripotent stem cells. These cells can be transformed into artificial tregs. This allows for a more efficient method of treg collection than painstakingly isolating tregs from the patient.
2. The inflammatory cytokine TNF-a is sensed by the TNFR 1 and 2 receptors on the cell surface. This notifies the treg that inflammation is on the rise and that the immune environment is unbalanced by sending a signal down a synthetic signalling pathway.
3. Said signaling pathway is seen above. The intracellular section of the receptors send a signal to a linker chain of proteins. These linkers brings the signal to the corresponding terminal of a Tobacco Etch Virus (TEV,) causing it to rejoin and regain function. The rejoined TEV cleaves the pathway at the TEV cleavage sites, freeing the two terminals of the dCas9 to rejoin and regain function.
4. With the aid of the two nuclear localization sequences on the dCas9, the rejoined dCas9-VPR is able to travel effectively to the nucleus and locate the IL-2 gene with aid from a matching guide RNA held within the dCas9. The three activator domains (VPR) then are able to activate the IL-2 gene, leading to IL-2 autocrine production.
5. When an excessive amount of IL- 2 is consumed by the treg, this can lead to decreased suppressive abilities and a reversed immune imbalance, which would only further CBI. The phosphorylation of the Ser473 binding site on AKT is utilized as an indicator that IL-2 production has become extreme.
5. The phosphorylation of the Ser473 triggers another custom protein chain, leading dCas9-VPR being tagged with the ubiquitin protein. This signals to surrounding proteins that the dCas9-VPR needs to be disposed of. By removing the dCas9-VPR, the IL-2 gene is once again silenced and treg growth is restored to baseline levels. This creates a seesaw-like system where the tregs balances between IL-2 silencing and activation based off of immune conditions.
dCas9 and CRISPR Explained
CRISPR dCas9-VPR is a gene editing technology that uses a dead Cas9 protein and guide RNA to locate a gene and activate it with three transcription activator domains.
The power of dCas9-VPR increases reliability in activating the IL-2 gene.
Additionally, its ability to reverse activation makes it suitable in the dynamic nature of the system. CRISPR technology as a whole would be utilize to implement the custom signaling pathways within the genome
Diagram of a simplified dCas9-VPR (neurotorium, 2024)