Skip to main content

Brain-Specific, Modular and Active RNA Therapeutics

Periodic Reporting for period 3 - B-SMART (Brain-Specific, Modular and Active RNA Therapeutics)

Reporting period: 2020-01-01 to 2021-06-30

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 integrate 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.
Highlights of the third 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.
Progress in Theme I
The work on the established lipid nanoparticles has resulted in our adoption of a clinically used formulation to be our default formulation. The current mRNA SARS-CoV2 vaccines that are very similar in composition underline that that remains the current state-of-the-art. At partner VIB, the immunization of Alpacas with receptors overexpressed at the cerebrospinal fluid barrier has been performed and a lead nanobody has been tested and validated. By understanding both the epitope on the CSF barrier to which the nanobody binds as well the trigger for the translocation to occur the value of this family of nanobodies is expected to increase. Methodology to track biodistribution of nanobodies, e.g. for assessment of biological barrier crossing, was developed and will be applied to ex vivo animal samples in the coming reporting period. This will improve the chances for future exploitation and commercialization.
Progress in Theme II
An important part of this work is devoted to being able to characterize the critical nanocarrier parameters that define activity. The use of advanced NTA modalities, like fluorescence, continues to show value for the characterization of nanocarriers developed in B-SMART, both for in-depth analysis and for support during development. In-depth characterization of the lead LNP formulation was performed, regarding size, composition and API quantification, showing good reproducibility and quality of the formulation. Stability studies were performed on the lead LNP formulation, demonstrating overall good stability of the components and formulation. Analytical endpoints included chemical composition, siRNA loading and size/size stability. A novel analytical method (FI-AF4) was developed and published in a peer reviewed journal.
Progress in Theme III
Theme III is devoted to performance of the nanocarriers regarding safety and efficacy in vitro and in vivo. The hemocompatibility test for the lipid-based nanocarriers has been completed. The reporter assay to identify early promise for activity is set up and running and has been completed for the DoE on the lipid-based nanoacarriers. In addition, we have access to/developed new reporter assays that are even more sensitive in detection of functional RNA delivery. These read-out systems are now available for the emerging and experimental systems.
Progress in Theme IV
Theme IV is designed to support the research and reach out to stakeholders. During the progress meetings, deliverables were presented and the progress in all work packages monitored. As an example of dissemination of knowledge to the scientific community, a peer-reviewed article on the value of FI-AF4 as new analytical tool for analysis of siRNA lipid nanoparticles was published. B-SMART members also joined important discussion for a for example the ETPN2020 “Workshop on translational issues” where especially the step form lab to market was extensively discussed.
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 scientists, where also Prof. Schiffelers is VP of pre-clinical R&D. In this company, DNA and RNA therapeutics are combined delivered by 20MED technology.
B-SMART logo