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Patrizia Tornabene, Paola Tiberi, Renato Minopoli, Alberto Auricchio; Gene therapy of Alström syndrome’s retinitis pigmentosa with multiple AAV vectors. Invest. Ophthalmol. Vis. Sci. 2017;58(8):1571.
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© ARVO (1962-2015); The Authors (2016-present)
Alstroöm syndrome (AS) is a rare monogenic disease inherited as autosomal recessive and characterised by retinitis pigmentosa (RP), among other features. AS is caused by mutations in ALMS1, which has a coding sequence (CDS) of 12.5 kb and encodes a large ciliary protein.Gene therapy with adeno-associated viral (AAV) vectors holds great promise for the treatment of many inherited retinal diseases including AS. The main obstacle for AS gene therapy is that AAV cargo capacity is limited to ∼5 kb. To overcome this, we split the ALMS1 expression cassette in 3 parts separately packaged in 3 independent AAV vectors that reconstitute ALMS1 upon co-administration and intermolecular recombination.
To develop AAV-mediated gene therapy for AS, triple AAV vectors were designed and the efficiency of triple AAV-mediated ALMS1 transduction was evaluate in vitro by infecting HEK293 cells with AAV2/2 vectors.To assess the percentage of photoreceptors (PR) co-transduction mediated by three independent vectors, we injected subretinally 4 week-old C57/BL6 mice (N=5) with single AAV8 vectors encoding either eGFP, DsRed or EBFP2 proteins. Then, the efficiency of triple AAV-mediated ALMS1 transduction was evaluate in vivo by subretinal injection of AAV2/8 in 4 week-old C57/BL6 mice (N=8). Western blot analysis was used to evaluate transgene expression. Retinal structure and function of the AS mouse model with and without delivery of AAV vectors was characterized by optical coherence tomography (OCT) and electroretinogram (ERG) analysis, respectively.
We show that triple AAV ALMS1 vectors reconstitute full length ALMS1 protein expression both in HEK 293 cells and mouse PR. We also observe that subretinal administration of 3 independent AAV vectors separately carrying the reporter eGFP, DsRed and EBFP2 transgenes results in about 10% PR co-transduction. Moreover, we show that the retina of the AS mouse model undergoes progressive outer nuclear layer thinning and reduction of electrical responses to light. Finally, a modest improvement, which was significant (p=0,047) albeit transient in the b-wave amplitude, was observed between 4 and 7 months of age in the Alms1−/− eyes injected with triple vectors compared to those injected with PBS.
Triple AAV vectors expand AAV transfer capacity in the retina up to 13Kb and delivery in the mouse model of AS results in transient improvement of electrical activity.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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