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Deliverable D2.1 “Regional Case Studies” summarises the work carried out under Task 2.1 (Set up of Quadruple Helix Multi-Actor Regional Groups) and Task 2.2 (Organisation of the multi-actor approach in case study regions) within Work Package 2. The deliverable documents the establishment of the project’s multi-actor framework at regional level, a key prerequisite for integrating ecological limits into bioeconomy strategies across Southern European regions. The report details the formation of the eight Multi-Actor Regional Groups (MARGs), comprising 92 representatives from the quadruple helix (policymakers, industry, academia, civil society), achieving a balanced gender composition. It also describes the preparatory interviews (35 in total) conducted to gather initial regional insights. A major focus is the organisation of the MARG Kick-Off Meetings held in all eight pilot regions in March 2025. These meetings introduced the BioINSouth project, validated preliminary regional findings, and initiated the co-creation of regional working plans for the development of the local BioINSouth HUBs. The KOMs mobilised strong stakeholder engagement, with 120 participants (86% attendance rate) and representation across all quadruple-helix categories. Key outcomes include the completion of a Regional Report and a Regional HUB Working Plan for each region, reflecting validated regional needs, challenges, and opportunities. The successful implementation of these activities confirms the effective establishment of the regional multi-actor structure. Overall, D2.1 provides the foundational input for the development and operationalisation of the regional HUBs (WP3) and will inform subsequent project activities, ensuring alignment with the diverse regional realities of Southern Europe. Further information about the project and its outputs is available at: https://www.bioinsouth.eu/.