Despite 6 products on the market and dozens in advanced phases of clinical development, in vivo gene therapy with vectors derived from adeno-associated viruses (AAV) still faces the major challenge of the limited vector cargo capacity (< 5kb). This prevents AAV application for treatment of inherited diseases caused by mutations in large genes. To address this, we have recently developed an AAV-based strategy relying on the use of short protein elements called inteins. These, when fused at the extremities of fragments of a large protein delivered to cells through AAV, can mediate joining of the fragments in a traceless manner, resulting in the reconstitution of the intact target protein. However, despite being extensively utilized as biotechnological tools in vitro, the in vivo application of inteins for therapeutic purposes comes with several limitations. These include: insufficient levels of full-length protein reconstitution for some applications; complex design; and production of undesired, potentially harmful by-products. NextGeneTx aims to expand the potential of AAV-inteins as an innovative in vivo biotechnological tool for therapeutic purposes, by addressing each of these limitations. To achieve this, we will leverage cutting-edge technologies to: i. engineer inteins with improved efficiency and greater design flexibility; ii. modulate intracellular trafficking of intein-containing protein fragments to enhance the efficiency and accuracy of full-length protein reconstitution; iii. develop strategies to reduce the production of unwanted by-products. We will test the therapeutic relevance of evolved AAV-inteins in both gene therapy and genome editing applications, using human retinal organoids and animal models of retinal diseases. The outcomes of NextGeneTx will define a highly adaptable, safe, and effective next-generation AAV-based approach to deliver large genes, thus significantly expanding the patient population that can benefit from in vivo gene therapy.

ProgRET will create a multidisciplinary and intersectoral European training network focusing on the mechanisms, diagnosis and therapy of dominantly inherited retinal diseases (IRD). IRD represent a major cause of blindness, affecting 350,000 people in Europe. IRD have long been considered incurable, however major advances have led to groundbreaking new treatments. Today, the most important challenges in the IRD field relate to an unsolved genetic diagnosis, unknown disease mechanisms and gene therapy development for autosomal dominant IRD (adIRD), representing 25–40% of all IRD cases. We have demonstrated an emerging role for splicing factors, structural variants and non-coding defects in patients with adIRD, and developed novel disease models and gene therapies for adIRD. ProgRET aims to dissect adIRD mechanisms using retinal stem cell and aquatic animal models, to advance adIRD diagnostics using a single-molecule multi-omics framework, and to develop innovative treatments based on RNA therapy and CRISPR-genome editing. These challenges will be tackled by integrating unique expertise and cutting-edge technology within ProgRET, including (multi-)omics, bioinformatics, functional genomics, RNA biology, gene regulation, stem cell technology, retinal organoids, animal models, genome editing and gene therapy. ProgRET will give Doctoral Candidates (DCs) unparalleled training opportunities in outstanding academic and industrial settings through training-by-research via individual research projects, secondments, and network-wide training sessions. All individual training and research activities will provide each DC with the necessary skills in academic and industrial research. ProgRET will make a career in both sectors attractive and improve their career prospects. Finally, our multidisciplinary network offers a unique opportunity to accelerate the understanding, diagnostics and therapeutics for adIRD in Europe, and to translate research findings to healthcare and society.