Viabilizando terapias génicas baseadas em vetores de AAV: expandindo o tamanho do genoma do vetor e melhorando a eficiência de entrega

Challenges in the state of the art: Several characteristics of the AAV vectors make them the vector of choice for in vivo applications: efficient tissue diffusion, its non-integrative safe profile, improved ability to transduce non-dividing cells and relatively low immunogenicity [1]. For these reasons, methodologies to overcome AAV vectors major disadvantage of small genome size is of high interest. However, AAV vectors transduce cells very poorly resulting in the clinic administration of exceedingly high doses. Enhancing AAV vector transduction efficiency is therefore essential for their effective and safe use. Objectives: how EVADE surpass current challenges EVADE advances the state of the art of AAV vectors by enabling the delivery of larger therapeutic transgenes. Currently dual AAV vectors provide low efficiency of full-therapeutic protein reconstitution. EVADE delivers novel highly efficient molecular technologies for re-constituting full-proteins through Cre mediated recombination. We will compare our Cre-based technology with protein trans-splicing technology that we recently established [2]. Thus, as specific objectives #1 and #2, this project generates 2 dual AAV vector systems: 1) a novel ground-breaking Cre/Lox DNA recombination methodology and 2) an advanced dual AAV protein trans-splicing for DMD and LCA10 (Fig.3). EVADE will work also at enhancing the vector itself, its immune profile and transduction efficiency. To improve AAV vector transduction efficiency beyond current state of the art EVADE explores the use of peptides: 1) targeting, 2) cell penetrating and 3) nuclear localization signal. Hence, EVADE project further specific objectives #3 and #4 are: to enhance AAV vector genome cassette and improve AAV transduction efficiencies. Important criteria to assess EVADE success in improving AAV vectors (objectives #1 to 4) is vector quality and potency that will be evaluated in vitro and in vivo. With that in mind we will establish methodologies: to assess vector capsid structural stability (objective #5), and to evaluate vector cellular trafficking and transduction efficiency (objective #6). EVADE translates the novel technologies to two clinically relevant diseases. AAV is the vector of choice for muscular and retinal dystrophies [13, 14]. However, the sizes of the therapeutic genes required to treat DMD and LCA10 collide with its limited DNA packaging capacity. Therefore, these disorders are ideal targets for dual AAV therapies. Taking advantage of consolidated expertise of F.Montanaro (UCL) and G. Silva (NMS) EVADE has as objectives #7 and 8#, to further elucidate and advance DMD and LCA10 gene therapies. In summary, EVADe will advance gene therapy state of the art by: 1. Developing innovative Cre/Lox DNA recombination AAV dual systems; 2. Advance intein protein trans-splicing AAV dual systems for DMD and LCA10 gene therapies; 3. Enhancing AAV vector genome cassette for improved therapeutic expression and reduced immunogenicity. 4. Improving AAV vector transduction efficiency. By implementing existing consortium knowledge and technical capability it will further advance towards a therapy proof of concept by: 5. Establishing a set of methods to evaluate AAV vectors potency in vitro: (i) immune assays; (ii) particle biophysical stability and (iii) vector transduction and trafficking assays; 6. Characterizing dual AAV vectors potency in vivo; 7. Advance DMD gene therapies; 8. Advance LCA10 gene therapies.