Inherited Retinal Diseases (IRDs) represent a major cause of blindness worldwide. Adeno-associated viral (AAV) vector-based gene therapies represent the most promising treatments. We aimed to develop gene therapies for gyrate atrophy of the choroid and retina (GA) and Usher syndrome type 1B (USH1B) retinitis pigmentosa. GA is characterized by ornithine aminotransferase [OAT, coding sequence (CDS) ∽1.3 Kb] deficiency. We demonstrated in vitro expression and activity of 3XFlag-tagged human OAT (hOAT-3XFlag). AAV vector carrying the hOAT-3XFlag expression cassette improved the structural retinal defects in the Oat-/- mouse model of GA. Bi-allelic mutations in the Myosin7A gene (MYO7A) (CDS ∽6.7 Kb) cause USH1B, the most common combination of inherited congenital deafness and blindness. We demonstrated effective delivery and expression of MYO7A in mice and pigs using dual AAV vectors. During AAV manufacturing, we found a contaminant vector resulting from recombination between two homologous sequences in the AAV vector containing the 5’ half of hMYO7A. This was removed by changing one of the two sequences while maintaining the same MYO7A expression levels in vivo. We selected three therapeutic doses of dual AAV-hMYO7A that rescue retinal defects in shaker-1 mice, a mouse model of USH1B. These doses will be translated in patients with USH1B. In the same mouse model, we confirmed biological potency of dual AAV-hMYO7A that will be used in the clinical trial. Overall, these studies offer promising results, paving the way for a gene therapy of GA and for the clinical translation of dual AAV vectors in USH1B subjects.
GENE THERAPY FOR GYRATE ATROPHY OF CHOROID AND RETINA AND FOR USH1B RETINITIS PIGMENTOSA / F. Dell'aquila ; tutor: A. Auricchio ; internal supervisor: I. Conte ; external supervisor: V. Marigo. Università degli Studi di Milano, 2021 Dec 13. 33. ciclo, Anno Accademico 2021. [10.13130/dell-aquila-fabio_phd2021-12-13].
GENE THERAPY FOR GYRATE ATROPHY OF CHOROID AND RETINA AND FOR USH1B RETINITIS PIGMENTOSA
F. Dell'Aquila
2021
Abstract
Inherited Retinal Diseases (IRDs) represent a major cause of blindness worldwide. Adeno-associated viral (AAV) vector-based gene therapies represent the most promising treatments. We aimed to develop gene therapies for gyrate atrophy of the choroid and retina (GA) and Usher syndrome type 1B (USH1B) retinitis pigmentosa. GA is characterized by ornithine aminotransferase [OAT, coding sequence (CDS) ∽1.3 Kb] deficiency. We demonstrated in vitro expression and activity of 3XFlag-tagged human OAT (hOAT-3XFlag). AAV vector carrying the hOAT-3XFlag expression cassette improved the structural retinal defects in the Oat-/- mouse model of GA. Bi-allelic mutations in the Myosin7A gene (MYO7A) (CDS ∽6.7 Kb) cause USH1B, the most common combination of inherited congenital deafness and blindness. We demonstrated effective delivery and expression of MYO7A in mice and pigs using dual AAV vectors. During AAV manufacturing, we found a contaminant vector resulting from recombination between two homologous sequences in the AAV vector containing the 5’ half of hMYO7A. This was removed by changing one of the two sequences while maintaining the same MYO7A expression levels in vivo. We selected three therapeutic doses of dual AAV-hMYO7A that rescue retinal defects in shaker-1 mice, a mouse model of USH1B. These doses will be translated in patients with USH1B. In the same mouse model, we confirmed biological potency of dual AAV-hMYO7A that will be used in the clinical trial. Overall, these studies offer promising results, paving the way for a gene therapy of GA and for the clinical translation of dual AAV vectors in USH1B subjects.File | Dimensione | Formato | |
---|---|---|---|
phd_unimi_R12094.pdf
Open Access dal 14/10/2023
Descrizione: Tesi Dottorato
Tipologia:
Tesi di dottorato completa
Dimensione
2.62 MB
Formato
Adobe PDF
|
2.62 MB | Adobe PDF | Visualizza/Apri |
Pubblicazioni consigliate
I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.