Delivering the messengerengineering nanoparticles for advanced mRNA therapies

Resumo

Messenger RNA has advanced to the forefront of innovative therapies in the past years. With the approval of COVID19 vaccines, mRNA has demonstrated its potential in vaccinology. Nevertheless, multiple biomedical applications can benefit from its advantages over other genetic therapies. Unlike DNA therapies, mRNA exerts its action in the cytoplasm, and, therefore, does not integrate into the nucleus of the host, a fact that is translated to higher safety profiles. For the same reason, the delivery of exogenous mRNA is a more efficient process, facilitating its use as a therapeutic. Furthermore, mRNA can be synthesized in the laboratory following straight forward techniques, which, at the same time are adaptable to industrial scale, including for GMP manufacturing. Importantly, mRNA sequence can be modified and adjusted to specific needs, such as emergency situations or personalized medicine. Nevertheless, important drawbacks have delayed mRNA therapies development since its discovery in 1961. Due to its physicochemical properties, this large and anionic biopolymer is impeded from functionality when naked administered, as it possesses a short half life, is hampered from crossing cellular membranes and prompts immunological reactions. As such, researchers have engineered ways to tackle these disadvantages. First, the modification of mRNA chemistry has permitted its translational application, by increasing in vivo potency without causing immunological responses. Second, the engineering of nanoparticles that protect, transport, and deliver mRNA to target cells has allowed its use as a therapeutic agent. The term nanoparticle is comprised in the term nanomedicine, which is described as formulations of various materials in the range of the nanometer scale. With several clinical products such as Doxil or Abraxane, nanomedicine has played a vital role in the success of mRNA therapies. In RNA space, the approval of Onpattro, a siRNA based therapy for the treatment of hereditary transthyretin mediated amyloidosis in 2018 has pivoted the way forward to todays most advanced delivery vehicle for mRNA, Lipid Nanoparticles. As of 2024, three mRNA therapies using LNPs have reached the market, and many others are in clinical development. The success of these particles relies on the discovery of ionizable lipids, lipids which are protonated only at acidic pH, as opposed to the traditional approach of using permanently charged lipids. As such, upon LNPs formulation at acidic pH, ionizable lipids permit the encapsulation of mRNA, however, after final formulation buffer adjustments, LNPs become neutral; overcoming the disadvantages of cationic charged particles. So far, the most advanced LNPs are composed of an ionizable lipid, cholesterol, DSPC and DMG PEG. Thus, the research of new lipids that can help push forward the development of more potent and specific or tailored particles is vital. As well, the use of new and more tolerable ionizable lipids and alternatives to PEG lipids are essential to grant higher safety profiles, especially important for several biomedical applications.In this context, the main objective of this thesis was to design and develop a technology, Sphingomyelin Nanoparticles, SNPs, which can effectively protect and deliver mRNA for biomedical applications, specifically inspired in a previously patented technology based on sphingomyelin SM and vitamin E VE, SNs. To this aim, the design, development and fully characterization of Sphingomyelin Nanoparticles is presented in Chapters I and II. Upon composition-based screenings and detailed study of the physicochemical properties, in vitro and in vivo performance, results showed that size, composition and particle concentration dramatically impact formulation performance. Additionally, our results indicate that SNPs can be modulated to modify organ tropism. On top of that, the design of totally biodegradable SNPs is also presented, overcoming the main drawbacks of current LNPs, and allowing for safer and more translational approaches. In Chapter III, the study of SNPs efficacy in a relevant therapeutic model is proposed, with a focus on an innovative application within immunooncology, which uses the liver as a factory of immunomodulatory proteins. The use of two previously reported SNPs, these results show remarkable tumor size decrease as well as tumor regression in half of the individuals of the treated group. In Chapter IV, the use of biodegradable SNPs for tissue reprogramming is studied. This novel application aims at tissue rejuvenation using mRNA upon delivering Yamanaka Factors. Results show how SNPs are capable of encapsulating all mRNA sequences an, for the first time, the delivery of Oct4 and Sox2 factors is reported.