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Targeted Amphoteric Carriers in ImmunoTherapy

Final Report Summary - TACIT (Targeted Amphoteric Carriers in ImmunoTherapy)

Executive Summary:

The last decade has been exciting for oligonucleotide-based drug discovery and development. The Nobel Prize was awarded to Mello and Fire for discovering RNA interference. There was also big investment from the Pharmaceutical industry to Biotechnology companies-pioneers in oligonucleotide therapeutics. High expectations were raised but further progress has been hampered by the issue of systemic delivery; siRNA oligonucleotides suffer from a poor pharmacological profile in vivo, whereas antisense DNA oligonucleotides have been shown to be effective for liver-specific targets only.

The network TACIT (Targeted Carriers for ImmunoTherapy) was formed by two academic (Biomedical Research Foundation of the Academy of Athens, University of Crete) and two commercial (Novosom AG, Cenix Bioscience) Partners to address this urgent need for effective systemic delivery systems by focusing on the administration of siRNA oligonucleotides to immune cells and inflamed sites. Novosom AG, a pioneering company on innovative lipid-based formulations, has developed a completely novel carrier system based on amphoteric liposomes. Its technology incorporates structural features with unique charge-reversal properties, which result into safety and performance advantages over the current best alternative carriers. Cenix Bioscience, a world leader in high throughput siRNA screening, has developed a proprietary peptide-based delivery approach, DARE. Biomedical Research Foundation of the Academy of Athens (BRFAA) possesses unique expertise on immune-mediated disease models and targeting of experimental therapeutics to immune cells and disease sites. Finally, University of Crete (UOC) has key know-how on molecular networks regulating inflammation and animal models of inflammatory disease. These Partners were brought together with the aim to significantly advance existing, and develop new, carrier technologies to develop novel improved and clinically relevant delivery modalities to cells of the immune system, and apply them for therapeutic oligonucleotide delivery to inflammatory diseases.

The programme consisted of six distinct but interdependent work packages involving the study of basic mechanisms controlling the stability, efficacy and safety of amphoteric liposomes (WP1), the development and selection of novel liposomal carriers, in some cases modified with cell specific ligands, for efficient systemic and cell-targeted delivery (WP2), the application of novel liposome and peptide carriers for the therapeutic delivery of ASOs, siRNA and microRNAs in mouse models of inflammatory disease (WP3-5), and the exploitation and dissemination of the study’s results (WP6). An extensive exchange programme of PhD and postdoctoral fellows between partners facilitating transfer of technology and know-how was implemented.

Implementation of the programme has been particularly fruitful with exciting results. Novel lipids with rational variations of their head group and lipid anchor chemistry were designed and synthesized. New liposomal carriers for systemic delivery of oligonucleotides were then developed by using new lipids and a quantitative structure-activity relationship (QSAR) methodology that predicts the stability and efficacy of liposomes previously established by Novosom. These were functionally tested for siRNA delivery in vitro, and specific highly effective formulations were identified. New liposomal carriers were further tested in vivo for their tropism to specific immune cell populations, such as macrophages and dendritic cells, potency for target knock-down and safety. Selected liposomal formulations were also examined for efficient miRNA and as-miRNA delivery, both in vitro and in vivo, and optimized liposomal formulations carrying miRNAs and anti-miRNAs targeting the let7/mir155 pathway were successfully applied for the treatment of LPS-induced endotoxin shock, DSS-induced inflammatory bowel disease and Pseudomonas auroginosa-induced lung injury in mice. Additionally, new liposomal formulations were developed for topical administration of siRNA to the lung. Selected formulations with targeted properties to the airway epithelium, or formulations with a broader distribution to both, airway and alveolar epithelium as well as lung macrophages were identified. Finally, an approach beyond liposomal formulations was also explored. This is based on the development and evaluation of novel peptide carriers, the DARE delivery technology of Cenix Bioscience, for the in vivo delivery of siRNA molecules. DARE delivery carriers were shown to efficiently deliver their cargo siRNA to macrophages and related cells, both in cell culture and in vivo in the ovalbumin-induced model of allergic asthma in mice. In addition to the identification of potent delivery vehicles, siRNA stability for in vivo experimentation was also addressed. Utilizing the expertise of Cenix Bioscience siRNAs were designed targeting a central signaling pathway in macrophage activation, this of Akt kinase, which were successfully applied in culture and in vivo in a model of aspiration-induced lung injury in mice. Furthermore, the program gave the opportunity to young scientists working in the academic environment to be exposed and obtain expertise in Biotech companies, which was transferred back to the academic labs. Expertise and technology on mouse models of inflammatory diseases was in parallel transferred from the academic partners to Cenix Bioscience.

Overall, new lipid-based and non-lipid carriers were developed through TACIT that can be effective for systemic or topical delivery of siRNA/miRNA oligonucleotides to immune cells and inflamed sites. As this has been a major obstacle hampering the development of siRNA-based therapeutics and siRNA-based target validation approaches, despite major investment from the pharmaceutical industry, TACIT is likely to have a broader impact to the wider field of oligonucleotide therapeutics. It will accelerate research in siRNA delivery systems and oligonucleotide therapeutics, it will strengthen the Partners’ portfolio and market position, and it will enhance the competitiveness of European research in a heavily US-dominated field. Ultimately, it will benefit the patients by providing new siRNA-based solutions for devastating medical conditions that cannot be confronted by more traditional approaches.