Next-generation direct reprogramming.

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.

Overcoming immune evasion from post-translational modification of a mutant KRAS epitope to achieve TCR-engineered T cell-mediated antitumor activity.

T cell receptor (TCR)-T cell immunotherapy, in which T cells are engineered to express a TCR specific for a tumor epitope, is a form of adoptive cell therapy (ACT) that has demonstrated promise against various tumor types. Mutants of oncoprotein KRAS, particularly at glycine-12 (G12), are frequent drivers of tumorigenicity, but also attractive targets for TCR-T cell therapy. However, MHC class I-restricted TCRs specifically targeting G12-mutant KRAS epitopes in the context of tumors expressing HLA-A2, the most common human HLA-A allele, have remained elusive despite evidence that an epitope encompassing the mutation can bind HLA-A2 and induce T cell responses. We report that post-translational modifications of the protein on this epitope may allow tumor cells to evade immunologic pressure from TCR-T cells. A lysine side chain-methylated KRAS G12V peptide, rather than unmodified epitope, may be presented in HLA-A2 by tumor cells and impact recognition by TCRs. Using a novel computationally guided approach to design TCRs, we developed by mutagenesis TCRs that recognize this methylated peptide, enhancing tumor recognition and destruction. Additionally, we identified TCRs with similar functional activity in normal repertoires from rare primary T cells by stimulation with modified peptide, clonal expansion, and selection. Mechanistically, a gene knockout screen to identify mechanism(s) by which tumor cells methylate or demethylate this epitope unveiled SPT6 as a demethylating protein that could be targeted to improve effectiveness of these TCRs. These findings highlight the role of post-translational modifications in immune evasion and suggest that identifying and targeting such modifications should facilitate development of more effective TCR-T cell therapies.