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Highly Efficient Conversion Of Human Ips Cells To Dopaminergic Neurons By Synthetic Modified Mrnas

Principal Investigator:

Ventral midbrain dopaminergic (mDA) neurons derived from human induced pluripotent stem cells (iPSCs) provide a unique cell resource for disease modeling, drug development and cell replacement therapy for Parkinson's disease (PD). Current strategies for generating transplantable iPSC-derived mDA neurons are still slow and variable. These strategies rely on using cell-extrinsic factors to modulate intracellular signaling and indirectly activate master transcription factors (TFs) (e.g. FOXA2, LMX1A, NURR1) that drive mDA neuron differentiation. Here, we propose to directly activate these master TFs by delivering synthetic mRNAs coding these TFs. In our preliminary studies, we successfully generated highly pure and functional iPSC-derived mDA neurons by sequentially delivering synthetic mRNAs coding four TFs (ATOH1, FOXA2, LMX1A and NURR1, referred to as “AFLN TFs”).

Our novel approach directly activates TFs to drive mDA neuron differentiation without relying on cell-extrinsic factors or virus-mediated gene delivery. Our preliminary studies support that this AFLN-mRNA-driven strategy is more rapid and efficient than commonly used methods for deriving mDA neurons from iPSCs. Here, we hypothesize that the sequential activation of AFLN TFs drives highly efficient conversion of iPSCs into mDA neurons by cooperatively activating genes essential for establishing and sustaining the mDA neuron identity. Our goal is to establish the first synthetic-mRNA-driven approach for generating highly pure and transplantable mDA neurons from iPSCs, and further translate this approach into products for producing iPSC-derived mDA neurons that serve as a reliable cell resource for disease modeling, drug testing and cell replacement therapy for PD.

  1. In Aim 1, building on our preliminary studies, we will optimize the AFLN-mRNA-driven strategy by modifying individual AFLN mRNA sequence to enhance TF protein stability and its activity in driving mDA neuron differentiation. We will extensively characterize the in vitro and in vivo functions of AFLN-induced mDA neurons. The goal of this aim is to establish a convenient and reliable approach for rapidly generating highly pure, functional and transplantable mDA neurons from both normal and PD-patient-derived iPSCs.
  2. In Aim 2, we propose to globally identify AFLN-activated genes during mDA neuron conversion, and further identify TFs that cooperate with AFLN TFs to drive mDA neuron differentiation. The goal of this aim is to identify and validate potential TFs that can be used to further improve the AFLN-mRNA-driven strategy. This project is highly innovative and translatable. We will establish the first synthetic-mRNA-driven method for efficiently generating iPSC-derived mDA neurons, which will provide a reliable cell resource for disease modeling, drug screening and cell replacement therapy for PD.

Bradley L. Schlaggar, M.D., Ph.D., Named President and CEO of Kennedy Krieger Institute

We’re thrilled to welcome Bradley L. Schlaggar, M.D., Ph.D., to the Kennedy Krieger family as our next President and CEO.
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