The overall objective of B-SMART is to create brain-targeted RNA-based nanomedicines for neurodegenerative diseases that are manufactured via a quality-by-design approach with precise nanoparticle characterization and specifications that meet the requirements for GMP scaling up and clinical translation.
ACTIVE MODULES – Brain-targeted RNA nanocarrier
The first theme of the B-SMART project focuses on the modules that together form the active nanocarriers. WP2 concerns the RNA payload, in WP3 the brain targeting ligands for attachment to the carrier surface are developed and WP4 is concentrated on the nanocarriers systems.
- WP2 – RNA Payload
The RNA payload, bearing the therapeutic activity, is a very attractive and effective therapeutic entity. It can easily be adjusted to target different mRNAs and thereby proteins and different mutations causing neurodegenerative diseases by adapting the sequence. Within WP2 three RNA sequences will be investigated and validated so that RNA sequences can be produced in a next phase.
At the same time, the RNA payload is a fragile molecule that can be destroyed by enzymes. In addition, difficulties can occur in reaching the site of activity: the cytoplasm of the diseased cell in the brain. The RNA payload is rapidly cleared by the kidneys and shows essentially no distribution towards the brain. And even if it would reach the brain, the molecule is relatively large and charged, making it virtually impossible to pass the cell membrane to reach the cytoplasm. This necessitates materials that protect and deliver the RNA – so-called RNA carriers.
- WP4 – RNA Carriers
The nanocarriers proposed within B-SMART should provide this protection and delivery. Four different nanocarriers will be investigated at three stages of development in WP4. They range from current established state of the art stable nucleic acid lipid particles, via emerging polymer-based responsive materials that possess attractive new properties, to exploratory biological extracellular vesicles that display remarkable functionalities which are not complete understood yet.
The nanocarriers can provide a protective barrier against enzymatic degradation. Due to nanoparticle encapsulation, renal clearance is prevented. In addition, they possess functional moieties that help RNA translocate across the cellular membrane. To arrive in the brain, the nanocarriers need to gain access which can be achieved by so-called targeting ligands on the surface of the nanoparticle.
- WP3 – Targeting Ligands
The nature of the brain-specific targeting ligand is dependent on the administration route. Three delivery routes will be explored in WP3 – local intracerebroventricular injection, intranasal administration and intravenous injection. Positive control ligands with demonstrated activity in vitro are available for all routes of administration.
A novel gateway to the brain, the brain-cerebrospinal fluid barrier, will be investigated for which new targeting ligands consisting of the variable domain of heavy chain only antibodies are developed and coupled to the nanocarrier surface. The targeting ligand should attach to surface receptors of barriers of the brain and permit translocation of the nanoparticle.
TOWARDS GMP – Manufacture & QC
The second project theme is focused on the scalable and reproducible manufacture of the complex systems. WP5 focusses on scalable manufacture of the nanocarriers using an easy to operate off-the-shelf microfluidic assembly system that will ensure quality-by-design: uniform nanocarriers across research sites, excellent control over the physicochemical parameters and linear scalability. For each of the nanocarrier systems appropriate quality control parameters have to be established in WP6 to ensure reproducible production within tight specifications. WP7 concerns the identification of the critical elements in translating the batch-by-batch laboratory scale production towards GMP.
- WP5 – Microfluidic Production
The self-assembly of the synthetic nanocarriers is driven by electrostatic complexation and hydrophobic interactions of multiple components. This self-assembly process is highly dependent on reaction conditions like volume, stirring speed and salt concentrations.
To remove operator variability and enable seamless scaleup and to allow robust manipulation of a single variable per experiment, microfluidics manufacture has been chosen at all partner research sites. This will result in well characterized nanocarriers in WP5 for which quality control specifications can be set ensuring activity compatible with GMP production.
- WP6 – Quality Control
Physicochemical characterization of the nanomedicine candidates demands special attention. In WP6 size and size distribution of the nanocarriers in relevant buffers and liquid phases will be investigated. Nanoparticle stability over time likewise needs to be verified.
Other critical parameters include loading efficiency of active RNA compounds and its release and stability in media. Release studies will be performed in complex biological media as a simulation of in vivo conditions. As the target proteins are relatively small, quantification of the intact proteins will be investigated as well.
- WP7 – Towards GMP
In WP7 microfluidic manufacture will be used to handle nanoparticle formulation aiming at the evaluation of each step from an industrial manufacturing point of view and the development of nanocarriers GMP production. The different processes to manufacture the modular nanocarriers will be evaluated, to verify their feasibility and scale up ability to production scale.
The critical parameters and quality specifications will be defined by a QbD approach to obtain a robust, repeatable and efficient production process. GMP production of nanocarriers will require continuous feedback between quality control and GMP production to monitor critical parameters, pharmaceutical specifications and stability, and to guarantee a successful GMP scaling up.
PERFORMANCE – Safety Aspects & Efficacy
The third project theme evaluates in vitro and in vivo safety and efficacy of the nanocarriers, where in vitro work will precede the in vivo investigations. WP8 investigates safety aspects and WP9 efficacy aspects, ultimately in preclinical models of Alzheimer’s disease and SBMA.
- WP8 – In vitro Compatibility and Tissue Distribution
Before embarking on efficacy studies, the safety of the nanomedicines will be tested in in vitro and in vivo assays in WP8 as this is an important criterion to enable industrial exploitation. Nanocarriers first come into contact with blood components, blood cells, and the cells of the immune system after intravenous administration.
Since these interactions could negatively affect their delivery to the target site or even worse, trigger systemic toxicity, it is vital to evaluate behavior of such particles in the blood before proceeding to in vivo studies. Additionally, the interaction with cells can lead to loss of viability. Tissue distribution profiles will highlight organs that are at highest risk for adverse effects.
- WP9 – Efficacy vitro & vivo
Ultimately, the efficacy in in vitro and in vivo models of neurodegenerative diseases should be demonstrated to show that the RNA is successfully delivered to the target cells in the brain and is affecting the disease process.
Thus, WP9 focusses on the characterisation of associated biological effects of brain targeted RNA carriers in vitro and in vivo using in vitro reporter and therapeutic assays and mouse models of neurodegenerative and rare diseases. The main focus is on Alzheimer’s disease (AD) and spinal and bulbular muscular atrophy (SBMA).
The nanocarriers that will be tested for therapeutic efficacy in vivo in WP9 will be selected based on their biological and physicochemical parameters identified in previous WPs. For example, these nanocarriers must have demonstrated biological activity in reporter cells and disease model cell lines (WP9), and shown their production via microfluidics is robust and reproducible (WP5) with desired physicochemical properties (WP6). Furthermore, the targeted nanocarriers must have displayed desired safety profile and CNS localisation (WP8) during previous testing. This balanced scorecard can feed back into WP2, WP3 and WP4 to optimize the design of the nanocarriers.