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Frank M Dyka, Laurie L Molday, Vince A Chiodo, Robert S Molday, William Hauswirth; Dual Adeno-Associated Virus Vector mediated gene therapy for autosomal recessive Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2019;60(9):3402.
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© ARVO (1962-2015); The Authors (2016-present)
The retina specific ATP binding cassette transporter A4 (ABCA4) is necessary for the clearance of all-trans-retinal from photoreceptor cells. Loss of this crucial function results in the accumulation of toxic bisretinoids, primarily N-retinylidene-N-retinylethanolamine (A2E). This ultimately leads to the Stargardt phenotype of increased autofluorescence and progressive RPE and photoreceptor cell loss. Adeno-associated virus (AAV) vectors have proven their utility for efficient gene transfer in the retina to a variety of cell types. However, the ABCA4 coding sequence (cds) of 6.8kb exceeds the payload capacity of a single AAV capsid of 4.8kb by far. AAV dual vectors have been shown to overcome this size restriction by splitting the cds between two vectors and packaging them in separate capsids. After co-infection of a cell the two vectored cDNAs recombine to reconstitute the full length cds. Here we present recent data on the effect of AAV dual vector mediated ABCA4 gene replacement therapy in an ABCA4 knock out mouse model of Stargardt disease
The human ABCA4 cds was cloned into AAV dual vector pairs. Each 5’ vector contains a strong ubiquitous promoter and the 5’ portion of the ABCA4 cds. Each 3’ vector contains the 3’ portion of the ABCA4 cds and a polyadenylation signal. Sets of ABCA4 knock out mice were injected subretinally with AAV dual vector pairs or the 5’ vector alone. Protein expression was detected by western blot and immunohistochemistry. The effect of the dual vector treatment was analyzed monthly by measuring the autofluorescence with a confocal scanning laser ophthalmoscope and the integrity of retinal layering by optical coherence tomography. A2E was quantified by HPLC relative to an external standard.
ABCA4 dual vectors produced full length ABCA4 protein both in-vitro and in-vivo and the protein localized correctly to the outer segments of photoreceptors. Fundus autofluorescence measurements showed that lipofuscin accumulation in dual vector treated versus 5’ vector only treated animals is significantly reduced. A2E quantification showed a parallel reduction.
AAV dual vectors are capable of delivering cDNAs exceeding the payload capacity of AAV with high efficiency and specificity. Our results indicate that AAV dual vector delivered ABCA4 has a significant effect on lipofuscin/A2E accumulation in the ABCA4-/- mouse model of Stargardt disease.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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