ULTRASTRUCTURAL PATHOLOGY AND BIOIMAGING LAB

Lipid-mRNA nanoparticles for tissue regeneration (130PED/2025)

PN-IV-INO-PED-2024-1  (2025-2027)

 

 

Ageing presents in immense burden on healthcare systems worldwide, therefore slowing and possibly reversal of ageing represent critical targets in modern biomedical research. This 2-year project is a collaboration between the Ultrastructural Pathology and Bioimaging Lab of Victor Babes National Institute of Pathology, Bucharest (IVB) and the company Blue Screen SRL. Together, we aim to test and validate custom-formulated LNP-mRNA compounds, encoding OSK factors, to be delivered to mammalian cells for epigenetic reprogramming and reversal of ageing, both in vitro and in vivo settings.

 

For this purpose, we will employ cutting-edge techniques, including epigenome sequencing, data-independent acquisition (DIA) technologies in mass spectrometry (SWATH-MS, label-free and library-free peptide mapping using neural networks and interference correction) for deep, quantitative-level proteome coverage, super-resolution fluorescence microscopy (STED) to track protein dynamics and high-resolution transmission electron microscopy (TEM) to analyze changes in cell morphology.
 

This work builds on existing preliminary data obtained by IVB and Blue Screen SRL, in collaboration. Specifically, Blue Screen has previously constructed stand-alone LNPs and has successfully delivered functional mRNAs to cultured cells using this encapsulation system. IVB validated the morphology of these LNP constructs and the purity of the suspension using TEM.
 

The project brings multiple elements of originality and innovation to the field of cellular reprogramming. To our knowledge, this is the first study to investigate partial cellular reprogramming processes through a time-course multi-omics approach, using Nanopore direct sequencing, unbiased DIA-MS methods and high-resolution imaging. If successful, this work would directly contribute to development of future therapies for ageing reversal and/or treatment of age-related diseases. The specific objectives of this project are:

1. Formulation of LNPs-OSK-mRNA for epigenetic clock reversal. At this stage we will construct LNPs for efficient and non-toxic delivery of OSK-mRNA to various types of somatic cells, aiming to successfully induce partial epigenetic reprogramming to a „youthful” state and reversal of the epigenetic clock (rejuvenation), without return to pluripotency.

2. In vitro rejuvenation of primary cell lines. At this stage we will simulate the ageing process in human primary cell lines through proliferation and successive passages, then, induce epigenetic rejuvenation by controlled OSK transfection of aged cells. DNA methylome changes and proteome quantitative dynamics will be tracked over time by Nanopore sequencing and MS, respectively. Cellular morphology and subcellular localization of key proteins will be tracked by STED and TEM.

3. In vivo evaluation of epigenetic rejuvenation. At this stage, we will evaluate the efficiency of LNP-OSK-mRNA constructs in a rat model, providing insights into the safety and practical applicability of mRNA-driven epigenetic rejuvenation in mammals, essentially representing a nonclinical proof of concept for these compounds. DNA methylome changes, proteome quantitative dynamics and cell morphology will be tracked in various tissues by Nanopore sequencing, MS and TEM.

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