Synthetic genetic feedback controller circuits to reprogram cell fate
PI: Domitilla Del Vecchio1;4
co-PIs: James J. Collins2;4;5;6, Thorsten Schlaeger7, and Ron Weiss2;3;4
1Department of Mechanical Engineering, MIT; 2Department of Biological Engineering, MIT
3Department of Electrical Engineering and Computer Science, MIT
4Synthetic Biology Center, MIT; 5Broad Institute of MIT & Harvard; 6The Wyss Institute
7 Stem Cell Transplantation Program, Boston Children's Hospital
PROJECT SUMMARY
The past decade has seen monumental discoveries in the stem cell ¿eld, with demonstrations that the fate of a
terminally differentiated cell, contrary to what was traditionally believed, could be reverted back to pluripotency or
directly converted to other differentiated cell types. All of a sudden, new approaches to regenerative medicine seem
within reach: lost or damaged cells could be replaced by patient-speci¿c reprogrammed cells, thus providing on-
demand, compatible, high-quality cells of any required type. To meet this vision, the scienti¿c community has made
tremendous efforts toward establishing robust and ef¿cient protocols for cell fate reprogramming. These protocols are
largely based on a priori ¿xed (pre¿xed) ectopic overexpression of suitable transcription factors (TFs), with the rationale
that this overexpression could trigger transitions among the states of the gene regulatory networks (GRNs) that take
part in cell fate determination. Yet, despite a decade of remarkable progress, the ef¿ciency of these protocols remains
low, the quality of produced cells is often unsatisfactory, and many potentially useful direct cell fate conversions still
seem impossible. These issues pose a formidable obstacle to the practical use of both human induced pluripotent stem
cells (hiPSCs) and transdifferentiated cells in regenerative medicine.
Arguably, our ability to accurately and precisely steer the concentrations of GRNs' TFs within desired ranges is critical
to the success of cell fate reprogramming. Unfortunately, current protocols based on pre¿xed TFs' overexpression have
not demonstrated this critical ability. To address this problem, we propose a completely new approach to cell fate
reprogramming in this project: we replace pre¿xed overexpression with feedback overexpression of TFs, which we
realize with an in vivo synthetic genetic feedback controller circuit. Within this circuit, the overexpression level is not a
priori ¿xed and is adjusted based on the discrepancy between desired and actual TF's concentrations. It therefore can
accurately and precisely control TFs' concentrations to desired values, independent of the endogenous GRN that also
regulates these TFs. Our research plan focuses ¿rst on hiPSC reprogramming as a test-bed for evaluating the bene¿t of
our approach and second on directed differentiation of hiPSCs into platelets as a directly clinically relevant application.
Speci¿cally, in AIM 1, we propose to systematically investigate the ef¿cacy of pre¿xed overexpression of pluripotency
TFs for hiPSC reprogramming. In AIM 2, we propose to construct and test the synthetic genetic feedback controller
circuits that implement feedback overexpression of a number of TFs concurrently. In AIM 3, we will leverage the
synthetic genetic feedback controller circuits for human hiPSC reprogramming and for directed differentiation of hiPSCs
into platelets. This project will result in substantially higher reprogramming ef¿ciencies, in cell products that more
closely resemble the target cell type, and in the future, in cell conversions that today seem not possible. More broadly,
our synthetic genetic feedback controllers will empower scientists and practitioners with a new tool to accurately control
the TFs' concentrations of any endogenous GRNs and, in particular, of those GRNs involved in cell fate determination.
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