Next-generation direct reprogramming

Abstract

Tissue repair is significantly compromised in the aging human body resulting in critical disease conditions (such as myocardial infarction or Alzheimer's disease) and imposing a tremendous burden on global health. Reprogramming approaches (partial or direct reprogramming) are considered fruitful in addressing this unmet medical need. However, the efficacy, cellular maturity and specific targeting are still major challenges of direct reprogramming. Here we describe novel approaches in direct reprogramming that address these challenges. Extracellular signaling pathways (Receptor tyrosine kinases, RTK and Receptor Serine/Theronine Kinase, RSTK) and epigenetic marks remain central in rewiring the cellular program to determine the cell fate. We propose that modern protein design technologies (AI-designed minibinders regulating RTKs/RSTK, epigenetic enzymes, or pioneer factors) have potential to solve the aforementioned challenges. An efficient transdifferentiation/direct reprogramming may in the future provide molecular strategies to collectively reduce aging, fibrosis, and degenerative diseases.

Keywords: aging; cardiac muscles; direct reprogramming; partial reprogramming; pioneer factors; signaling; skeletal muscle; transdifferentiation.

FIGURE 1

A summary of transdifferentiation fibroblast into skeletal, cardiac muscles and neurons. We summarize here various cocktails of transcription factors and their combination with small molecules or micoRNAs their lineage specific trans differentiation such as Dopaminergic neurons (Vierbuchen et al., 2010; Pfisterer et al., 2011a; Pfisterer et al., 2011b), GABAminergic (Lee et al., 2020; Bruzelius et al., 2021); Noradregenic neuron (Ainsworth et al., 2018); Myofibre (Davis et al., 1987; Tapscott et al., 1988; Weintraub et al., 1991); iMPCs (Qabrati et al., 2023) and Cardiomyocyte (Ieda et al., 2010; Qian et al., 2012; Song et al., 2012; Wada et al., 2013). Image created by Biorender.

FIGURE 2

Signaling cascades augments transdifferentiation through gene expression. Various receptor tyrosine kinases (RTKs) or receptor Serine/Threonine Kinase (RSTKs) have been targeted in various direct reprogramming. RTKs such as VEGFR, FGFR, Insulin receptors acts through (A) JAK-STAT signalling, activated Janus Kinase downstream of the receptor phosphorylates STAT3. STAT3 dimers dependent gene expression is initiated. (C) MAPK pathway- RasGTP results in further phosphorylation of Raf, MEK and ERK1/2; phosphorylated ERK1/2 phosphorylate transcription factors (TF such as JUN,FOS, MYC, etc.) that activates their target genes. (B) PI3K pathway Activated PI3K downstream of the receptor phphorylates AKT. pAKT phosphorylation repress transcription factors such as FOXO (shown as example), BAD, TSC2, p27 while activates other transcription factors such as MDM2 and eNOS. RSTKs such as BMPR and TGF- $\beta$ R phosphorylates (D) R-Smads: Smad1/Smad5/Smad8 and Smad2/Smad3 respectively. These phosphorylated R-Smads forms complex with co-Smad4 that translocate in the nucleus resulting in transactivation of target genes. (E) TGF- $\beta$ R can also acts through JAK-STAT pathway (shown by dashed arrow). Image created by Biorender.

FIGURE 3

Novel approaches in transdifferentiation. First, the fibroblast receptors can be targeted with designed miniproteins that can enhance the reprogramming. Second, modified mRNA based reprogramming where transgene free expression of lineage specific transcription factors can reprogram the fibroblast to specific lineage (Qabrati et al., 2023). Third, the usage of epigenetic tools such as CRISPR mediated opening of promoters of specific pioneer transcription factor genes that are silenced in the fibroblast (Liu et al., 2018; Jiang et al., 2022). Fourth, combination of a pioneer factor with lineage specific transcription factor can open chromatin landscape allowing efficient reprogramming to multiple lineages. Image created by Biorender.