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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 1 - B-SMART (Brain-Specific, Modular and Active RNA Therapeutics)

Teaser

Summary

The overall objective of B-SMART is to create brain-targeted RNA-based nanomedicines for neuro-degenerative diseases, such as Alzheimerâ€™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.

Work performed

Highlights of first reporting period
The work is progressing according to plan and in some cases is ahead of schedule in achieving its objectives. For the established frontrunner, lipid-based nanomedicines that we make in Theme I, we have 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 (Theme III). In fact, the lipid-based nanocarriers have progressed so well through the manufacture, experimental design, quality control and in vitro stages that they have already entered in vivo experiments within this reporting period. In this particular set-up, we have prepared lipid-based nanocarriers loaded with miR-132 that have been applied intranasally in a murine model of Alzheimerâ€™s disease.

Progress in Theme I
The established lipid carriers progress fast through the development process. As foreseen, the emerging nanocarriers, i.e. nanogels, nanocapsules and nano-emulsions require more work to progress to this stage. For these emerging carriers, prototypes have been made that are currently in in vitro testing. For the experimental type of nanocarriers, i.e. the extracellular vesicles, experimental protocols have been validated.
For the targeting ligandscandidates have been identified. These candidates are proteins known as nanobodies. Nanobodies are engineered from a peculiar kind of antibody that is found in animals from the camel-family, such as llamas, alpacas and camels.

Progress in Theme II
At all sites the same microfluidic manufacture equipment has been acquired and is currently operational. An important result that was anticipated by using the same equipment for manufacture was that we would obtain the same particles at every institute, provided that the raw materials are the same. This has proven to be true. An important part of this work is devoted to being able to characterize the critical nanocarrier parameters that define safety and therapeutic effects.

Progress in Theme III
Theme III is devoted to performance of the nanocarriers both regarding safety and efficacy in vitro and in vivo. The hemocompatibility test for the lipid-based nanocarriers has been completed according to the protocols generated by the Nanomedicine Characterization Laboratory (Frederick, USA). These tests can now be used for the emerging and experimental nanocarriers. Therapeutic in vitro and in vivo assays for Alzheimers disease and Spinal Bulbar Muscular Atrophy are running.

Progress in Theme IV
Theme IV is designed to support the research and reach out to stakeholders. We have quickly fulfilled the administrative requirements to start the project, implemented the Consortium Agreement, and organized the kick-off meeting.In the following months, dissemination materials were prepared, including a website, folder and a project identity ppt template. During the progress meetings the first deliverables were presented and the progress in all work packages monitored. We were joined by outside experts to sharpen our focus and defined critical issues for our progress. As outreach to the general public we have prepared a short movie that was uploaded on social media platforms like YouTube. The LinkedIn post of the movie by the coordinator was seen by more than 4000 viewers within 10 days after posting.

Final results

In Theme I we have worked on three different types of carriers: established, emerging and exploratory. Especially for the emerging and exploratory vehicles we have moved beyond the state-of-the-art and demonstrated the promises and challenges of these formulations. For all three types we expect to be able to deliver a full package of characteristics of their safety and efficacy for brain delivery of siRNA.
The targeting ligands that we have identified are currently tested for activity; these could have important impact also beyond B-SMART, as access to the brain is an inherent challenge for all drugs that need to work in this tissue. A nanobody targeting ligand that could bring also these types of drugs to the brain would have widespread applications. In addition, these nanobodies could be important research tools.
Within Theme II the benefits of microfluidic manufacture for nanomedicine production are becoming apparent. This new production paradigm is an important asset for the field as it improves reproducible and homogenous particle preparation.
In Theme III we have shown the first safety and efficacy data. Together with the other themes we expect to arrive at an attractive data package for further industrial exploitation towards the end of the project. Active outreach during conferences has already provided industrial interest in our work.
Beyond B-SMART we foresee that our work contributes to clinical translation and industrial exploitation. The positive experience with microfluidic manufacture and the integration with the EUNCL infrastructure provides a case-study for a more rapid development of nanomedicines.