3 Daniel Simão, Catarina Pinto , Paulo Fernandes , Christopher J. Peddie , Stefania 4 Piersanti , Lucy M. Collinson, Sara Salinas , Isabella Saggio , Giampietro 5 Schiavo, Eric J. Kremer , Catarina Brito, Paula M. Alves 1,2 6 7 iBET Instituto de Biologia Experimental e Tecnológica, Apartado 12, 2780-901 8 Oeiras, Portugal 9 Instituto de Tecnologia Química e Biológica, Universidade Nova de Lisboa, A v. da 10 República, 2780-157 Oeiras, Portugal 11 Cancer Research UK London Research Institute, Lincoln's Inn Fields Lab oratories, 44 12 Lincoln's Inn Fields, London WC2A 3LY, United Kingdom 13 Dipartimento di Biologia e Biotecnologie “Charles Darwin”, Università di R oma La 14 Sapienza, Piazzale Aldo Moro 5, 00185 Rome, Italy 15 Institut de Génétique Moléculaire de Montpellier, CNRS 5535, 1919 Route de Mende, 16 34293 Montpellier,, France 17 Université Montpellier, 34293 Montpellier, France 18 Istituto Pasteur Fondazione Cenci Bolognetti, Università di Roma La Sapienza , Stanza 19 3-13, Piano 2, Piazzale Aldo Moro no5, 00185 Rome, Italy 20 Istituto di Biologia e Patologia Molecolari del CNR, Università di Roma La Sapi enza, 21 Piazzale Aldo Moro 5, 00185 Rome, Italy 22 Sobell Department of Motor Neuroscience and Movement Disorders, Institute of 23 Neurology, University College London, Gower Street, London WC1E 6BT, UK 24 25

Gene therapy is a promising approach with enormous potential for treatment of neurodegenerative disorders. Viral vectors derived from canine adenovirus type 2 (CAV-2) present attractive features for gene delivery strategies in the human brain, by preferentially transducing neurons, are capable of efficient axonal transport to afferent brain structures, have a 30-kb cloning capacity and have low innate and induced immunogenicity in preclinical tests. For clinical translation, in-depth preclinical evaluation of efficacy and safety in a human setting is primordial. Stem cell-derived human neural cells have a great potential as complementary tools by bridging the gap between animal models, which often diverge considerably from human phenotype, and clinical trials. Herein, we explore helper-dependent CAV-2 (hd-CAV-2) efficacy and safety for gene delivery in a human stem cell-derived 3D neural in vitro model. Assessment of hd-CAV-2 vector efficacy was performed at different multiplicities of infection, by evaluating transgene expression and impact on cell viability, ultrastructural cellular organization and neuronal gene expression. Under optimized conditions, hd-CAV-2 transduction led to stable long-term transgene expression with minimal toxicity. hd-CAV-2 preferentially transduced neurons, whereas human adenovirus type 5 (HAdV5) showed increased tropism toward glial cells. This work demonstrates, in a physiologically relevant 3D model, that hd-CAV-2 vectors are efficient tools for gene delivery to human neurons, with stable long-term transgene expression and minimal cytotoxicity.

Helper-dependent adenovirus vectors (HDVs) are safe and efficient tools for gene transfer with high cloning capacity. However, the multiple amplification steps needed to produce HDVs hamper a robust production process and in turn the availability of high-quality vectors. To understand the factors behind the low productivity, we analyzed the progression of HDV life cycle. Canine adenovirus (Ad) type 2 vectors, holding attractive features to overcome immunogenic concerns and treat neurobiological disorders, were the focus of this work. When compared with E1-deleted (ΔE1) vectors, we found a faster helper genome replication during HDV production. This was consistent with an upregulation of the Ad polymerase and pre-terminal protein and led to higher and earlier expression of structural proteins. Although genome packaging occurred similarly to ΔE1 vectors, more immature capsids were obtained during HDV production, which led to a ~4-fold increase in physical-to-infectious particles ratio. The higher viral protein content in HDV-producing cells was also consistent with an increased activation of autophagy and cell death, in which earlier cell death compromised volumetric productivity. The increased empty capsids and earlier cell death found in HDV production may partially contribute to the lower vector infectivity. However, an HDV-specific factor responsible for a defective maturation process should be also involved to fully explain the low infectious titers. This study showed how a deregulated Ad cycle progression affected cell line homeostasis and HDV propagation, highlighting the impact of vector genome design on virus–cell interaction.