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Frank M Dyka, Vince A Chiodo, William W Hauswirth; Adeno-associated virus dual vector-mediated gene therapy for ABCA4 Stargardt Disease. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4497.
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© ARVO (1962-2015); The Authors (2016-present)
ATP binding cassette transporter A4 (ABCA4) is a flippase localized in the rim region of photoreceptor outer segments. It is involved in the clearance of all-trans-retinal from the disks. Loss of this function leads to the accumulation of N-retinylidene-N-retinylethanolamine (A2E) in the RPE, a major component of Lipofuscin and ultimately the Stargardt Disease phenotype. Mutations in the ABCA4 gene are associated with the most common form of recessive Stargardt Disease. As a monogenic disease it is a viable target for gene replacement therapy. Lentiviral vectors are suboptimal for the transduction of differentiated cells like photoreceptors. In contrast, Adeno-associated virus vectors have shown great potential for gene transfer due to their low immunogenicity and their ability to infect dividing and post-mitotic cells equally efficiently. However, the payload capacity of AAV is limited to 4.8 kb. Hence the large size of the ABCA4 coding sequence of 6.8 kilobases, make it impossible to deliver the transgene in a single capsid. To overcome this drawback dual vectors were developed where the coding sequence is split between two vectors and packaged in separate capsids. After co-infection the two halves come together through homologous recombination or nonhomologous end joining to reconstitute the full length coding sequence. Here we present data on AAV dual vector mediated gene expression of ABCA4 and its effect on A2E accumulation in vivo in an ABCA4 knock out mouse.
The human ABCA4 coding sequence was cloned into AAV dual vector pairs, where one vector contained the promoter and the 5’ portion of the cDNA sequence and the second vector contained the 3’ portion and a polyA signal. ABCA4 knock out mice were injected with AAV dual vector pairs and retinal function was measured by electroretinography followed by lipofuscin/A2E autofluorescence employing a confocal scanning laser ophthalmoscope.
Expression of full length ABCA4 from AAV dual vectors was detected in vitro and in vivo. In vivo measurements revealed a marked reduction in A2E accumulation by fundus autofluorescence in injected versus uninjected eyes of ABCA4 knock out mice.
cDNAs exceeding the payload capacity of AAV can be expressed with high efficiency and specificity using dual AAV vectors. Our results show that AAV dual vector delivered ABCA4 can have a significant effect on A2E accumulation.
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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