# Synthetic Genetic Controller Circuits for Transcription Factor-Directed Differentiation

> **NIH NIH R56** · MASSACHUSETTS INSTITUTE OF TECHNOLOGY · 2024 · $600,000

## Abstract

PROJECT SUMMARY
 The ultimate goal of this project is to create synthetic genetic circuits that accurately control the level of cell fate-
specific transcription factors (TFs) autonomously in response to cell state changes. The underlying hypothesis is that
the level and timing of expression of critical TFs dictates the efficiency of cell conversion protocols and the quality of
produced cells. Here, we focus on the differentiation of human induced pluripotent stem cells (hiPSCs) into hemogenic
endothelial cells (HECs) from which all hematopoietic stem and progenitor cells (HSC/HPCs) arise. Unfortunately, cur-
rent methods to derive definite HECs (dHECs), which have the potential to produce adult-type lymphoid cells and HSCs,
remain not only inefficient but are also difficult to execute and scale, and, as a consequence, exhibit high degrees of
variability in outcomes between different labs, hiPSC lines, and even between replicate experiments.These problems
hamper analysis of the underlying developmental processes and pose formidable obstacles to clinical translation of
hiPSC-derived blood cell products since ensuring the safety and cost-effectiveness of the product necessitates high
differentiation efficiency and consistency. Prior work has demonstrated that SCL (S), LMO2 (L), GATA2 (G), and ETV2
(E) TFs together are sufficient to convert hiPSCs-derived mesoderm to dHECs and that efficient forward programming
requires discovery and subsequent implementation of both optimal expression levels and timing for each TF. Yet, con-
ventional methods for TF-mediated cell fate programming rely on indiscriminate overexpression without any control on
cellular TF levels. This is largely due to our inability to precisely control TF levels at user-defined values during cell
fate programming, and this limitation has prevented discovering optimal trajectories and subsequently enforcing them.
Here, we propose synthetic genetic controller circuits that overcome this hurdle. Specifically, in Aim 1, we create ge-
netic circuit designs that set TF levels and use them in an efficient in vitro differentiation protocol to discover the optimal
combination of S, L, G, E levels and timing. In Aim 2, we develop a new circuit architecture, based on TET1-enabled
positive feedback, to prevent epigenetic silencing of our genetic circuits once we deliver them to hiPSCs. In Aim 3,
we make our genetic controller circuits enforce autonomously the optimal SLGE TF levels found in Aim 1 in response
to the hiPSC-to-mesoderm transition. We achieve this by a new autocatalytic ADAR-based RNA sense-and-respond
system, which senses the mesoderm marker Brachyury (TBXT) and enforces user-defined TF levels in response to
it. We expect that this process, by being autonomous as opposed to manual and by enforcing optimal TF trajectories,
will result in a more efficient, repeatable, and robust hiPSCs to dHECs conversion protocol, thereby helping fill the
gap to clinical translation. Although in this...

## Key facts

- **NIH application ID:** 11117441
- **Project number:** 1R56EB036090-01
- **Recipient organization:** MASSACHUSETTS INSTITUTE OF TECHNOLOGY
- **Principal Investigator:** Domitilla Del Vecchio
- **Activity code:** R56 (R01, R21, SBIR, etc.)
- **Funding institute:** NIH
- **Fiscal year:** 2024
- **Award amount:** $600,000
- **Award type:** 1
- **Project period:** 2024-08-06 → 2026-01-31

## Primary source

NIH RePORTER: https://reporter.nih.gov/project-details/11117441

## Citation

> US National Institutes of Health, RePORTER application 11117441, Synthetic Genetic Controller Circuits for Transcription Factor-Directed Differentiation (1R56EB036090-01). Retrieved via AI Analytics 2026-06-12 from https://api.ai-analytics.org/grant/nih/11117441. Licensed CC0.

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