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Brain-Specific, Modular and Active RNA Therapeutics

Periodic Reporting for period 2 - B-SMART (Brain-Specific, Modular and Active RNA Therapeutics)

Reporting period: 2018-07-01 to 2019-12-31

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 second 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. As envisioned, with the microfluidic manufacture set-up using the same settings on the machine results in the same nanoparticles irrespective of the operator or the environment. This is an important step with regard to quality control and also with regard to the safety of the nanoparticles reproducible therapeutic efficacy. We have made important progress on the emerging carrier materials and 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. This nanobody has important commercial and scientific value that will also be exploited outside of B-SMART. Partner VIB has taken the initiative to patent this finding. In addition, we have made important progress with the emerging nanoparticles. In an attempt to circumvent the brain barriers encountered after intravenous administration, the nose-to-brain route was explored. These rationally designed nanocomplexes were produced using the microfluidics-based technique leading to (i) a unimodal distribution with a tunable mean size, (ii) the capacity to highly associate (100%) and protect RNA from degradation, (iii) the ability to preserve its physicochemical properties in biorelevant media and prevent the premature RNA release. In vivo experiments in a mouse model of Alzheimer's disease provided evidence of a statistically significant delivery of a potentially therapeutic miRNA mimic in the hippocampus area and its further effect on two mRNA targets, following its intranasal administration. This illustrates the emerging material’s potential for delivery of RNA.
Progress in Theme I
The work on the established SNALPs has resulted in our adoption of a clinically used formulation to be our default formulation. Adaptations to the orginal design of the emerging nanocarriers were introduced. For the targeting ligands, lectins and RVG-peptides, versions that can be coupled to the nanocarriers have been prepared based on S-acetylthioacetate maleimide chemistry as well as sortase. 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. Because of the wide application potential of this nanobody the finding has been patented.
Progress in Theme II
We are setting up an interlaboratory comparison test where we use a cheap lipid formulation to test proof of concept of the robustness of production and characterization while minimizing the financial costs of these tests. Partner 20Med demonstrated that the nanogel coating with hyaluronic acid was compatibel with microfluidic manufacture and resulted in improved characteristics compared to other production methods; the same holds true for the production of the enveloped nanocomplexes from partner USC. An important part of this work is devoted to being able to characterize the critical nanocarrier parameters that define activity. Partner SINTEF AS, as part of the European Nanotechnology Characterization Laboratory (EU-NCL) infrastructure, has developed protocols for the physicochemical analyses. Partner Malvern is adapting their particle characterization equipment especially for the most challenging, experimental, nanocarriers: the extracellular vesicles.
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. Also an outlook for the next period was presented. During the Utrecht meeting we were joined by staff from our microfluidic manufacture platform. They provided important insights into the performance of their system as well as the new innovations that have been introduced. They also provided access to some industrial contacts that are of interest to the consortium.
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. The plan was to include one Roche representative as a member of the B-SMART Industrial & Exploitation Advisory Board. Unfortunately, despite prolonged attempts and intense negotiations, the closure of a confidentiality agreement fell through. Still we will continue to be in contact to see if circumstances change that may allow to reactivate this collaboration. After a presentation at the Utrecht Brain Conference, an initial contact with the CEO of a start-up company developing immune therapies for Alzhiemer’s disease was established. We are now in the process of identifying opportunities for collaboration. Two partners are progressing in their valorization of research results: Partner VIB has filed a patent and partner Malvern developed a protocol for labelling extracellular vesicles (EV).