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Brain-Specific, Modular and Active RNA Therapeutics

Periodic Reporting for period 4 - B-SMART (Brain-Specific, Modular and Active RNA Therapeutics)

Reporting period: 2021-07-01 to 2022-06-30

The overall objective of B-SMART is to create brain-targeted RNA-based nanomedicines for neuro-degenerative diseases, such as Alzheimer’s disease. Often, such nanomedicines are highly complex compared to conventional therapeutics. They are often assembled in small quantities under variable conditions and the success is strongly dependent on the experience of the operator. This makes reproducible activity difficult. Therefore, in B-SMART, we try to make sure that the nanomedicines are manufactured via a quality-by-design approach with precise nanoparticle characterization and specifications that meet the requirements for scaling up and clinical translation.
To achieve this, we integrated four project themes:
I. To design nanomedicines consisting of 3 modules (a) an RNA payload, (b) the nanocarrier material and (c) a brain-specific targeting ligand.
II. To ensure that we can make the nanomedicines of Theme I using an easy to operate microfluidic assembly system that will ensure high quality nanomedicines that are reproducible and fit for clinical investigations. In microfluidic manufacture, the nanomedicines form by mixing two fluids in a cartridge. The characteristics of the nanomedicine that comes out of this process are dictated by a couple of simple parameters (like fluid flow rate and flow rate ratio) that will result in the same nanomedicines irrespective of operator and laboratory.
III. To evaluate the high quality nanomedicine of Theme II regarding pre-clinical activity of formulations regarding safety and therapeutic efficacy.
IV. To optimally support the research and promote industrial exploitation, we coordinate activities in Theme IV.
Conclusion: We have successfully assembled the nanomedicines, based on the different nanocarrier designs (established lipid nanoparticles, emerging carriers, exploratory extracellular vesicles). The lipid nanoparticles were selected as frontrunner applications, whereas new designs for the emerging carriers led to improvements of nanogels and nanoemulsions. The work on the lipid nanoparticles mirrored developments around these carrier systems in the world as a lipid nanoparticle formulation with siRNA targeting transthyretin (patisiran) and mRNA vaccines encoding coronavirus spike protein (comirnaty, spikevax) were approved during the B-SMART lifetime. We explored the reproducible manufacture. We have established that microfluidic manufacture is excellently suited to make lipid nanoparticles across sites. Given the questions around accessibility of this technology, we have also investigated rapid mixing protocols. The lipid nanoparticles prepared by both technologies were indistinguishable regarding physicochemical characteristics and biological performance. Based on these results we have finalized the design for a GMP facility for manufacture of RNA nanomedicines that can serve as service provider for production of formulation that are ready for clinical testing.
The lipid nanoparticles demonstrated an excellent safety profile and where highly active in their organ of natural tropism i.e. the liver. Silencing in the brain could be observed but was not efficient enough to expect a therapeutic benefit.
Highlights of the 4th reporting period: The work is progressing according to plan. For the established frontrunner, i.e. lipid-based nanomedicines, we made sure that we can make exactly the same nanoparticles in different laboratories and also with different manufacturing technologies i.e. microfluidic mixing and rapid mixing. This is important as the production of the mRNA-based SARS-CoV2 vaccines has shown that rapid mixing appears to have become the new state-of-the-art.
Both on a QC as well as biological performance level, the lipid nanoparticles produced with both technologies were indistinguishable.
We have extended the work on the targeting nanobody that would enable translocation across the blood cerebrospinal fluid barrier. We established that the preferred 2HF042 nanobody can deliver a small peptide (i.e. neurotensin) over the blood cerebrospinal fluid barrier and functionally deliver this into the brain and are trying to determine the epitope to which it binds and determine what causes the translocation effect to occur. Several similar nanobodies do bind but lack the translocation phenomenon which is important to understand to be able to fully exploit.
In addition, we have made important progress with the emerging nanoparticles that for several application appear to outperform the lipid nanoparticles as state-of-the-art. Understanding the basis for this performance and specific conditions at which this occurs allows to sketch a path forward for future academic and industrial applications.

During the final period of the project, results have been validated and steps to be taken to enable exploitation after the project end have been prepared. The partners have analysed the results with a view to the TRL levels expected at the end of the project, IPR management and suitable exploitation routes. In summary, it can be said that we have
- identified nanobodies that are transported across the blood-cerebrospinal fluid barrier in mice and monkeys and can deliver a small payload (peptide). This can be exploited as a research reagent, especially in combination with the highly similar nanobodies that were identified that lack translocation capabilities. These can be immediately exploited.
- identified nanobodies and nanobody motifs for medical use. The identified nanobody sequences and motifs that dictate translocation will be patented. VIB will take the lead in this and establish the best route of exploitation, be it through a spin-off, or licensing opportunities.
- identified improvements in the composition of 20MED nanogels and coating technologies for the nanogels that will be pursued by 20MED.
- identified a novel composition of nanoemulsions that can carry and deliver siRNA (USC). USC will take the lead in protecting the invention.
- incorporated the technical expertise on the different nanocarriers (lipid nanoparticles, nanogels, nanoemulsions, nanoenvelopes, extracellular vesicles) into the design of equipment that can help R&D characterization of nanoparticles (MALVERN). This equipment is currently ready for beta-testing at partner sites (e.g. UMCU).
- made the design of a GMP plant (IBI) that can serve a service provider for the manufacture of GMP/GCP material for late preclinical and early clinical studies. This is important for the expansion of the field with many RNA formulations currently entering this stage of development.
- established a network within and beyond the consortium to initiate follow-up projects, several of these have already been established, such as H2020 EXPERT (UMCU, Eurice, SINTEF), Nanospresso-NL (SINTEF, UMCU)
In the OpenData initiative, we aim to collaborate with two H2020 projects dealing with nanosafety issues. We have been approached by Roche, Switzerland with interest in the project that will be followed up upon when the patent for the nanobodies has been finalized as there may be overlap with in house developed technology. SINTEF joined a discussion group of EMA/FDA on RNA therapeutics. Two of the Utrecht PhD students that are finishing their theses have joined Nanocell Therapeutics as scientist, where also Prof. Schiffelers is part-time employed as VP pre-clinical R&D. In this company DNA and RNA therapeutics are combined delivered by 20MED technology.

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