Development of a novel PTD-mediated IVT-mRNA delivery platform for potential protein replacement therapy of metabolic/genetic disorders

IVT-mRNA is now being explored as a prominent therapeutic and vaccination platform for a variety of acute and chronic disorders. IVT-mRNA-based therapy is also applied to protein replacement therapy, cancer immunotherapy, cell reprogramming, and genome editing. Recently, COVID-19 vaccines have also been developed using the IVT-mRNA technology. Improvements in IVT-mRNA delivery have had a major impact on accelerating the advance of IVT-mRNA technology toward the clinic. Formulations that protect the IVT-mRNA from harmful nucleases and promote their cellular uptake combined with improvements to the IVT-mRNA molecules are critical advancements for making IVT-mRNA a viable therapeutic. Structural and functional modifications of IVT-mRNA can reduce the enzymatic degradation and improve the translation efficiency of the IVT-mRNA. The modifications include modified nucleosides, anti-reverse cap analogs (ARCAs), polyadenylation of the 3′ end, replacing of adenylate/uridylate rich elements (AREs) with 5′ untranslated regions of more stable mRNAs (like the murine 5′ UTR of the β-globin gene, as well as the human 3′ UTR of the α-globin or β-globin genes), and a Kozak sequence at the 5′ untranslated region.

In a new study from the Aristotle University of Thessaloniki, scientists Androulla Miliotou, PhD, Professors Lefkothea Papadopoulou, Ioannis Vizirianakis, Asterios Tsiftsoglou and Efthimia Vlachaki (also at Hippokrateion General Hospital), as well Professors Ioannis Pappas at University of Thessaly and George Spyroulias at University of Patras, developed a novel PTD-IVT-mRNA platform to deliver IVT-mRNA inside cells effectively and safely. The IVT-mRNA was conjugated with a selected Protein Transduction Domain (PTD). PTD/nucleic acid complexes can be generated either using electrostatic self-assembly for non-covalent bonding or chemical ligands for covalent bonding of the peptide / PTD with the mRNA cargo. PTD-IVT-mRNA platform is a potential therapeutic approach for disease models that are genetically aberrant and protein defective. The innovative platform was exploited to deliver the IVT-mRNA of SCO2 and β-globin, into primary SCO2 deficient human fibroblast cells, derived from a patient with fatal infantile cardio-encephalomyopathy and cytochrome c oxidase (COX) deficiency, and β-globin deficient hematopoietic cells, respectively. This molecular design model overcomes some of the challenges concerning translation, transduction, and stability of IVT-mRNA and demonstrated efficient translation of the desired proteins using transduced IVT-mRNA.

The research team successfully generated the plasmid vector that served as the in vitro transcription template, followed by the production of the corresponding IVT-mRNA and conjugated IVT-mRNA via a novel chemical reaction with a selected PTD. The complex was confirmed using band-shift assay and NMR spectroscopy. Structural ability and transducibility of the complex were verified at both RNA and protein level. PTD conjugation with the IVT-mRNA allowed the efficient cellular uptake of IVT-mRNA and subsequent translation into the desired proteins: a) in SCO2 deficient human fibroblast cells, transduction of the PTD-IVT-mRNA is mediated by clathrin-dependent endocytosis, leading to increased SCO2 translation; β) in β-thalassemia bone marrow cells from three individual patients, the transduction of PTD-IVT-mRNA led to increased translation of the β–globin in the recipient cells. The original research article is now published in the Journal, Molecular Theory Nucleic Acids.

Summarizing, the authors devised a newer IVT-mRNA delivery platform for protein therapy for the treatment of monogenetic or metabolic diseases, comprising IVT-mRNA and a selected PTD (PFVYLI). This delivery platform serves as an efficient alternative for previous delivery models, such as lipid-based IVT-mRNA carriers. The PTD is employed as an alternative to other gene therapy tools with low cytotoxicity and increased efficacy in transducing proteins and IVT-mRNA into the cells. The authors were successful in demonstrating the efficacy of the novel delivery platform in two monogenic disorders including a mitochondrial disorder (with SCO2 mutations) and β-thalassemia (β-globin mutations).

In a nutshell, the authors conducted an in-depth assessment of the developed method for the conjugation of a PTD to the IVT-mRNA (Greek patent:1010063; International publication number: WO 2021/094792 A1 / 20.05.2021) to keep the nucleic acid sequence stable, despite being exposed to RNases, and to deliver the IVT-mRNA for specific applications. The delivery formulation protects the IVT-mRNA cargo in the extracellular milieu and promotes its cellular uptake, release into the cytoplasm and efficient translation. It is hoped such continuous advancement of innovative delivery methods will pave the way for IVT-mRNAs to be tested in clinical trials to provide missing or defective proteins due to genetic disorders or in cases where the delivered protein imparts a therapeutic effect.