title: Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.
abstract: Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4-/- mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5'-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4-/- mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1.
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Ivana
authors.initials: I
authors.lastname: Trapani
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Elisabetta
authors.initials: E
authors.lastname: Toriello
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Sonia
authors.initials: S
authors.lastname: de Simone
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Pasqualina
authors.initials: P
authors.lastname: Colella
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Carolina
authors.initials: C
authors.lastname: Iodice
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Elena V
authors.initials: EV
authors.lastname: Polishchuk
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Andrea
authors.initials: A
authors.lastname: Sommella
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Linda
authors.initials: L
authors.lastname: Colecchi
authors.affiliation: Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Second University of Naples, 80121, Naples, Italy.
authors.forename: Settimio
authors.initials: S
authors.lastname: Rossi
authors.affiliation: Eye Clinic, Multidisciplinary Department of Medical, Surgical and Dental Sciences, Second University of Naples, 80121, Naples, Italy.
authors.forename: Francesca
authors.initials: F
authors.lastname: Simonelli
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Bologna 40064, Italy and.
authors.forename: Massimo
authors.initials: M
authors.lastname: Giunti
authors.affiliation: Department of Veterinary Medical Sciences, University of Bologna, Bologna 40064, Italy and.
authors.forename: Maria L
authors.initials: ML
authors.lastname: Bacci
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy.
authors.forename: Roman S
authors.initials: RS
authors.lastname: Polishchuk
authors.affiliation: Telethon Institute of Genetics and Medicine (TIGEM), Pozzuoli 80078, Italy, Medical Genetics, Department of Translational Medicine, Federico II University, Naples 80131, Italy auricchio@tigem.it.
authors.forename: Alberto
authors.initials: A
authors.lastname: Auricchio
mesh_terms: D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D060433:Administration, Ophthalmic; D000818:Animals; D000229:Dependovirus / Q000235:genetics*; D004195:Disease Models, Animal; D005260:Female; D015316:Genetic Therapy / Q000379:methods*; D005822:Genetic Vectors / Q000008:administration & dosage; D057809:HEK293 Cells; D006801:Humans; D008268:Macular Degeneration / Q000151:congenital* / Q000235:genetics / Q000628:therapy; D051379:Mice; D012160:Retina / Q000378:metabolism; D000080362:Stargardt Disease; D013552:Swine; D020079:Terminal Repeat Sequences; D019076:Transgenes
chemical_list: C056475:ABCA4 protein, human; D018528:ATP-Binding Cassette Transporters