Novel dual-vector delivery system for Stargardt disease gene therapy

The American biotechnology company Applied Genetic Technologies Corporation (AGTC), specialized in the development of gene therapies for rare diseases, has just announced Stargardt disease as its new preclinical gene therapy program in ophthalmology.

Stargardt disease characterized by a progressive loss of central vision affects one in 8,000-10,000 individuals and is the most common form of inherited macular degeneration. The first symptoms usually appear during childhood or adolescence and most cases are caused by mutations in the ABCA4 gene, which encodes a transporter protein present in the photoreceptor cells of the retina. Loss of ABCA4 function results in the accumulation of toxic bisretinoids in the photoreceptor outer disk membranes. Bisretinoids are formed during phototransduction and their accumulation leads to the degeneration of RPE and photoreceptor cells and eventually to the loss of central vision.

Efficient therapies for Stargardt disease are not yet available, although several drugs to reduce the accumulation of toxic substances in the retina and the pigment epithelium are now under study (read our blog entry on 06/25/19). A promising therapeutic strategy would be gene addition therapy, based on the transfer into retinal cells of a correct copy of the ABCA4 gene, to rescue the lack of function of the mutated gene. This gene therapy approach generally uses a type of vectors known as adeno-associated viruses (AAV), but AAVs have a limited packaging capacity of about 4.5 kilobases (kb) and the ABCA4 cDNA sequence is 6.8-kb long.

The challenge is therefore to devise a strategy to introduce larger genes in the retina, such as ABCA4. AGTC has just announced a dual-vector gene therapy system developed by researchers from the Department of Ophthalmology at the University of Florida, led by William Hauswirth. This method consists of dividing the coding sequence of ABCA4 into two fragments that are then packaged separately into two AAV vectors. Once inside the cell, the two DNA fragments recombine and reconstitute the full ABCA4 coding sequence, from which a functional ABCA4 protein can be synthesized.

Hauswirth team has just published a study in the journal Human Gene Therapy, in which they have tested, first in culture cells, three different dual vector systems for ABCA4 (overlap, transplicing and hybrid) and showed that the latter yields higher levels of ABCA4 expression. Next, researchers tested the therapy in an animal model of Stargardt disease, the knockout mice Abca4-/- lacking the ABCA4 gene. After subretinal injection of dual AAV into KO mice, full-length ABCA4 protein correctly localized to photoreceptor outer segments. Finally, researchers found that the gene therapy resulted in a reduced accumulation of lipofuscin/A2F, a toxic bisretinoid characteristic of Stargardt disease.

AGTC will continue the preclinical development of this therapy and has announced proof-of-concept data in non-human primates. According to William Hauswirth, “AGTC’s demonstration that this approach is safe and results in ABCA4 protein expression in non-human primates following subretinal injection of their optimized vectors is an important milestone in advancing gene therapy for Stargardt disease toward the clinic.”

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