Idea: Test Oct4 transcription factor alone to see if it can safely roll back the epigenetic clock of human cells in vitro while preserving their differentiation.

Explanation: Several studies report that Oct4 is the initiating reprogramming factor. For example, see the graph on the right.

Also, the following papers support the idea that Oct4 begins the process of epigenetic rollback:

• http://www.sciencedirect.com/science/article/pii/S0962892413002237
• http://onlinelibrary.wiley.com/doi/10.1002/stem.765/full
• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5291097/

It has been previously shown in several studies that OSKM induction (where O stands for Oct4) lowers epigenetic age gradually.

For example, this fact has been demonstrated in human fibroblasts in the following preprint:
https://www.biorxiv.org/content/early/2018/03/31/292680

Having discovered this gradual property of epigenetic reprogramming, we can now be cautiously optimistic that we will manage to find a safe therapeutic window (such as marked by a yellow box in the graph on the right) - i.e. a period of safe epigenetic rollback when the methylation profile of a cell has been returned to a younger state but the cell has not lost its functional characteristics (i.e. a skin cell remains a skin cell rather than gets dedifferentiated into a pluripotent cell).

We believe that we can now use a similar experimental design to confirm that Oct4 alone can also roll back the epigenetic clock, and do so more safely than OSKM. We would also like to find an optimal dosing regimen that provides lasting rejuvenation without risk of dedifferentiation.

We are encouraged in our hypothesis not only by the papers cited above but also by the graph on the right from another study, which shows that Oct4 alone lowers epigenetic age of human fibroblasts.

Our intention is thus to construct an Oct4 vector under a Tet-On induction system and then use it to transfect human cells. We would then induce Oct4 expression for different consecutive days and study the changes in methylation that occur.

Milestones:
1. Oct4 vector construction under Tet-On control. Time: 3 months.
2. Cell cultivation and transfection. Time: 2 months.
3. Oct4 induction and methylation sequencing for cells at various time points. Time: 2 months.

Budget: We estimate the budget for this experiment at $15 000 USD.

As some of you might know, we have teamed up with Gabriel Licina and his crew for this experiment. Recently, we finalized the design of our custom combined vector carrying the rtTA gene and the Oct4 gene under the Tet-On system. Here is our final map:

Once the vector build is complete, we will check that it works by transfecting human dermal fibroblasts and measuring Oct4 expression levels with and without Dox.

After that, we will carry out the main experimental procedures of differential Dox induction in human dermal fibroblasts (from a donor aged 40-50) transfected with our vector, collect resulting cells and ship them off to Zymo for methylation clock analysis.

Our intended dosing protocol is as follows: induce Oct4 expression in transfected HDF for 1,2,3 or 5 consecutive days via Dox. On the final day of each induction cycle, harvest part of the culture to be sent for sequencing. Use the remaining part of the culture for relaxation/washout experiments to see if methylation returns to its original configuration: i.e. stop Dox induction and harvest cells after 2,6,12 days after Dox withdrawal.

Stay tuned for further updates!