Job Description

The goal of this project is to create a composite platform for the delivery of modified RNA (modRNA), both by direct intramuscular and intracoronary injection. The project will focus on modRNA delivery into cardiac cells, in vitro, and into mice and pigs myocardium, in vivo. Typically, we will test transfection in ischemic models such as myocardial infarction (MI) or heart failure (HF). It’s been shown that spontaneous uptake of nucleic acids is insufficient; therefore, many viral and non-viral delivery vehicles have been developed to deliver nucleic acid. In the case of mRNA, two main categories of delivery agents were developed: delivery via physical disturbance of the membrane, to allow mRNA entry, and delivery by cationic particles via cellular endocytosis.

The first approach includes methods such as electroporation, gene gun, high-pressure injection and ultrasound that have limiting transfection abilities with low cell survival. The second approach includes the design of lipid or polymer-based cationic nanoparticles that interact with the nucleic acids via electrostatic interactions, thus enabling complex formation and spontaneous transfection agent formation. Design of these particles is based on delivery vehicles, previously used to transfect plasmid DNA. Even though some of these particles have shown the ability to transfect mRNA, single stranded mRNA strongly binds to cationic polymers, compared to plasmid DNA. Thus, efficient cationic polymers used for plasmid DNA transfection may not be suitable for efficient mRNA delivery. Also, recent reports raise serious concerns regarding cationic delivery systems for systemic use due to their aggregation ability, serum instability and cytotoxicity.

To overcome the barriers described above, especially with polymer-based cationic nanoparticles, we suggest the use of anionic nanoparticales complex for the delivery of modRNA. ModRNA technology is a novel and highly efficient approach used both in vitro and in vivo, to deliver one or more genes to a heterogeneous population of cells. Exogenous unmodified mRNA that enters the cell via the cell membrane is recognized by endosomal Toll-like receptors 7/8. This process inhibits protein translation and activates the innate immune response, ultimately leading to apoptosis of the hosting cell. ModRNA is synthesized by substituting ribonucleotides with naturally modified ribonucleotides. The use of these modified ribonucleotides results in changing the secondary structure of the synthesized mRNA, which prevents the Toll-like receptor from recognizing the modRNA and therefore permitting its translation to a mature protein by the ribosomal machinery without eliciting immune response or compromising the genome. ModRNA can be introduced to different cell types, as well as to both dividing and undividing cells with high efficiency, leading to immediate and high levels of protein expression in a pulse like kinetic.
The project will be to find a way to deliver the modRNA to cells and tissues using anionic nanoparticales. The anionic nanoparticales can be used for in vivo delivery as it was shown that the complex has stability under destabilizing conditions of heparin and plasma proteins presence. Also, cytocompatibility studies have shown no negative effect on the cells in terms of viability and most importantly expression of oxidative stress-related genes. Internalization studies using anionic nanoparticales and siRNA presented 96% efficiency in gene silencing.

In conclusion, anionic nanoparticales have shown very promising results for encapsulation and delivery of siRNA in vitro, with great potential for in vivo applications. Candidate will fabricate anionic nanoparticles, encapsulating modRNA by electrostatic interactions, bridged by calcium ions and then test their ability to efficiently delivery of modRNA into different target cells and tissues.

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