Spoke 8 - RNA Genetherapy

A major obstacle in developing and applying nucleic acid therapeutics and gene therapies lies in the lack of efficient delivery technologies that, besides preserving their fragile structure in the biological environment, offer efficient delivery of the therapeutic cargo to the target of interest. Spoke 8 is designed to reshape the delivery strategies for nucleic acid therapeutics taking into account the biological barriers to overcome and ensuring a low toxicity profile. Spoke 8 includes over 100 participants from 10 Italian institutions, who hold established expertise in drug delivery technologies. The collaboration extends to five private pharmaceutical companies, fostering a strategic partnership to bridge the gap between basic research and clinics.

The primary goal of Spoke 8 is to pioneer a new era of drug delivery technologies and expedite the transformation of theoretical therapeutic concepts from vertical Spokes into tangible pharmaceutical products. Central to this objective is the advancement of knowledge in pharmaceutical nanotechnologies. This includes exploring innovative functional materials, developing strategies for engineering nanoparticles for precise delivery into the body, creating human-relevant models for assessing delivery efficiency, and developing novel manufacturing technologies.

Close cooperation with industrial partners will ensure the practicality and regulatory compliance of the delivery platforms proposed on the bench, which will drive the translation of theoretical concepts into impactful pharmaceutical products.

PROGRAM

Spoke 8 research program holds three distinct Work Packages (WPs), each with specific tasks led by field experts. The primary focus of WP 8.1 is to create an extensive library of advanced delivery platforms using both non-viral and viral technologies. This entails developing innovative lipid and polymeric nanoparticles, viral vectors, and vesicles (microalgal, protozoan, and autologous vesicles) that facilitate precise delivery of nucleic acids to target cell populations and diseased areas within the body. Activities also encompass research on microneedle technologies for mRNA skin delivery and liver transfection to overcome organ rejection. Quality-centric bench preparation of nanoplatforms will maintain constant control over Critical Quality Attributes (CQAs) such as size, polydispersity, shape, charge, physical and chemical stability, and release kinetics. These attributes significantly affect pharmacokinetics, tissue biodistribution, cell uptake, and transfection efficiency. Specific quality check protocols will validate lab data, ensuring progression only for compliant nanoplatforms. The delivery platforms developed will be available to vertical Spokes 1-5 to evaluate their potential as novel therapeutic candidates.

WP 8.2 focuses on developing complex biological models (2D/3D, human organoids, organ-on-chip, in embryo models) that mirror disease complexities, which aim to reduce animal usage to assess delivery efficiency and toxicity. This includes integrating omics into nanotechnology to evaluate interaction with biological components, alongside live cell imaging through optical nanoscopy for assessing endosomal escape. This strategy aims to refine and select promising candidates developed in the project and to expedite Spoke 9 preclinical studies.

WP 8.3 aims to manufacture pharmaceutical prototypes that meet the Investigational New Drug quality standards in the early developmental phase. This involves implementing scalable current Good Manufacturing Practice (cGMP) compliant technologies like microfluidics and supercritical fluids, as well as transitioning from small laboratory batches to large industrial volumes, and selecting excipients from final formulations. Nanoplatform production lines found under cGMP conditions enable the progression of robust prototypes for preclinical animal studies and address challenges of their scalability early on. Activities also include evaluating formulation-container compatibility and exploring the feasibility of obtaining final powder formulations. We are developing specific Artificial Intelligence (AI) solutions and monitoring tools to ensure sample quality control between labs. AI is used to elaborate results by linking coherent nanoprototype properties to biological outcomes.

SPOKE FLAGSHIPS

Spoke 8 will expand its research infrastructures to meet the needs of its ambitious program, which challenges and intends to reshape the delivery strategies on interactions with the biological environment and regulatory constraints.

Implementing a research platform dedicated to omics science intends to expedite nanoparticle development by deciphering their interactions with living systems, aiding the design of targeted variants, predicting nanoparticle-biological interactions and toxicities, and tailoring treatments based on individual responses. The platform includes an infrastructure for evaluating the delivery efficiency in advanced biological models.

The creation of a High Throughput Formulation Screening platform intends to accelerate the discovery and optimization of nanoparticles by rapidly assessing many formulations and parameters. The platform utilizes automated systems that rapidly and in parallel produce nanoparticles. This includes robotic liquid handling systems for precise and consistent reagent mixing, nanoparticle formulation, and purification. The platform will be integrated with high-throughput analytical techniques to characterize nanoparticles rapidly. This permits screening a wide range of conditions quickly to identify the best-performing compositions, generate nanoparticle libraries, and identify correlations between nanoparticle characteristics and biological activity.

Lastly, Spoke 8 is setting up a Nanoprototype Lab designed to manufacture pharmaceutical formulations that meet the criteria of current Good Manufacturing Practices (cGMP) guidelines. The area is equipped with Class A, B, and C cleanrooms for sterile product production and scalable industrial-level equipment. The unclassified area is devoted to evaluating the Critical Quality Attributes of the final prototypes through validated procedures.

CROSS-SPOKE ACTIVITIES

Spoke 2. Exosomes for RNA delivery to solid tumors.
Spoke 3. Cationic liposomes with non-peptidic oligo cationic lipidic enhancers (OCEs) for siRNA delivery in neurodegenerative applications.
Spoke 4. Surface-engineered LNPs for precision delivery of RNA to the musculoskeletal system, CaP lipoic nanoparticles for siRNA cardiac delivery, exosomes for specific siRNA targeted to metabolic pathways in diabetic nephropathy, vesicles for miRNA delivery in liver fibrosis, metabolome profiling of nanoliposomes with LAV-BFIB4 for cardiovascular disease treatment.
Spoke 5. Camouflaged nanoplatforms for Type 1 Diabetes
Spoke 6. Double-targeted nanoparticles for the delivery of miRNA to breast cancer cell lines, nanoimaging platforms for selecting and optimizing the most suitable technologies for programmable RNA editing within cells.
Spoke 9. Regulatory issues raised by the clinical protocol of patient-derived extracellular vesicles, human tumor biopsy collection, and organoid production.

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PROGRAM

WHO WE ARE

SPOKE FLAGSHIPS

CROSS-SPOKE ACTIVITIES