(170b) Poly(β-amino ester) Terpolymer Nanoparticles As Delivery Vehicles for mRNA and DNA

Introduction: A host of human diseases are caused by missing or aberrant proteins. Effective treatment of the root cause of these maladies therefore requires replacement of such proteins. The most common means of protein replacement therapy is simply to administer the protein itself, but protein stability concerns often limit the effectiveness of this strategy. Alternatively, one can deliver nucleic acids coding for the aberrant protein. Nucleic acids are more easily stabilized than proteins, and can be condensed and encapsulated within delivery vehicles which can facilitate their effective in vivo transport. To date, DNA has been the most-studied nucleic acid for protein replacement therapy due to its relatively high stability and low immunogenicity when compared to mRNA. However, mRNA does not pose a risk of insertional mutagenesis, and it need only reach the cytoplasm to be effective. Additionally, recent innovations in mRNA synthesis have yielded more stable and potentially less immunogenic nucleic acids, further increasing the interest in delivering mRNA for protein replacement therapy. Thus, there is now a significant interest in designing materials to deliver mRNA in vivo. Previously, our lab reported the synthesis of poly(β-amino ester) terpolymers (PBAE terpolymers), copolymeric materials capable of electrostatically condensing DNA into nanoparticles that can additionally be formulated with other hydrophobic excipients. In this work, we utilize PBAEs to deliver mRNA and DNA in vitro and in vivo. 

Materials and Methods: PBAE terpolymers which had previously been synthesized for DNA delivery were synthesized via step polymerization, with synthesis conditions optimized for purification in diethyl ether to remove excess monomer. PBAE terpolymer nanoparticles were formed by nanoprecipitation via charge-based interactions with nucleic acids, as well as hydrophobic interactions between the PBAE terpolymer and a lipid-anchored PEG (PEG-lipid). Particle serum stability was assessed by doping nanoparticles with fetal bovine serum and measuring optical absorbance of the particle/serum solution over a 2-hour span at 37°C. In vitro nanoparticle efficacy was assessed by formulation PBAE nanoparticles with luciferase-encoding mRNA or DNA and transfecting HeLa cells, with cellular luminescence measured as an indicator of luciferase protein production. In vivo experiments were performed by delivering nanoparticles intravenously to C57/BL6. In a typical example, a mouse was dosed with 0.5 mg/kg of luciferase-coding mRNA loaded into a PEGylated PBAE terpolymer, and luminescence was quantified after 24 hours using an IVIS imaging apparatus.

Results and Discussion: We demonstrate that PBAE terpolymers can also be used to encapsulate and deliver mRNA both in vitro and in vivo. Specifically, we show that mRNA-loaded terpolymer nanoparticles can be co-formulated with (PEG-lipid) to improve the serum stability of terpolymers, as well as to increase in vivo potency. Nanoparticles that were stabilized in vitro were also successful in delivering nucleic acid to the lungs following intravenous delivery to mice, although biodistribution experiments revealed that nucleic acid localization was spread throughout major organs. Additionally, we utilize PBAEs as a platform to compare the protein production of mRNA and DNA. Our data shows that certain aspects of PBAE structure, especially the diacrylate used to form the polymer backbone, influence whether the material will be more successful for mRNA or DNA delivery. We further compare the role of other factors, such as polymer molecular weight and formulation parameters, for PBAE-mediated mRNA and DNA delivery. Furthermore, we plan on taking advantage of the in vivo potency of PEGylated PBAE nanoparticles to compare the potency of DNA and mRNA-based protein production.

Conclusions: In total, our research has shown that PBAE terpolymers are a therapeutically-relevant material for nucleic acid delivery. PBAEs are also useful given that they can deliver both mRNA and DNA. Their ability to deliver both nucleic acids successfully, along with their facile synthesis, can be leveraged to investigate chemical and formulation parameters that favor delivery of one nucleic acid over the other, as well as protein production afforded by both nucleic acid types in vivo.