Researchers at the Laboratory for Retinal Gene Therapy, Department of Ophthalmology, University Hospital Zurich, Switzerland, have reported a novel treatment approach aimed to expand the repertoire of gene transcripts beyond the AAV low genome packaging capacity of 4.7–5.0 kb. Their publication, published in Nature Communications (2023, 14:6578), have presented a dual AAV strategy to split larger therapeutic genes packaged across two AAVs and then reconstituted the functional gene in the host cells. While the research is at an early stage, a reconstituted example, using a full-length ABCA4, demonstrated the principle transfecting the dual strategy after intravitreal injection in a model of Stargardt disease. Following the report, the researchers stated that the technology was capable to lead a reconstitution of the ABCA4 gene with “correct localization of a therapeutically relevant large gene upon intravitreal injection”.
The new methodology, called “REVeRT”, present a dual AAV vector technology based on reconstitution via mRNA trans-splicing. The approach was applied to Stargardt disease (STGD1), an autosomal recessive retinal dystrophy with a prevalence of about 1/10,000, estimating that there are ~500,000 patients with the disorder worldwide. STGD1 is caused by mutations in the photoreceptor-specific ABCA4 gene which encodes the adenosine triphosphate-binding cassette, subfamily A, member 4. The ABCA4 gene encodes a transporter protein within retinal photoreceptor cells facilitating the active transport of potentially toxic retinoid compounds removing toxic by-products from the visual cycle. Research have previously reported that over 900 unique variants have been identified in ABCA4-related retinopathies and 50% of the variants are among missense mutations. The ABCA4 gene is located on chromosome 1p22.1 with a size of 7,328 nucleotides mRNA, encoding a 2,273-amino acid protein with a molecular mass of 255,944 Da. The gene has 7 transcripts (splice variants), 287 orthologues, 11 paralogues and is associated with 9 phenotypes. The clinical phenotype for the disease varies widely from mild visual complaints up to 50 years age or more, to severe visual impairment occurring at a young adolescence age with advanced retinal degeneration.
The present Swiss team stated that, “REVeRT is flexible in split site selection and can efficiently reconstitute different split genes in numerous in vitro models, in human organoids, and in vivo”. The approach of reconstitution at the AAV genome level have different versions on using homologous recombination and/or concatemerization however, similar efficiencies may also be used as split inteins – protein analogs of introns and exons found in relevant DNA and RNA sequence. In particular, researchers found their approach had at least three advantages over split inteins from a therapeutic perspective: (i) they show higher flexibility in the split site selection to support reconstitution; (ii) the efficiency of reconstitution appeared less dependent on the position of the split site and does not require proper protein folding prior to reconstitution, and; (iii) It does not generate bacterial proteins, which subsequently reduce the risk of immune responses in treated individuals. In conclusion, the researchers commented that, “We have obtained preliminary experimental data suggesting improvement of retinal degeneration and function in a Stargardt mouse model using the dual REVeRT vector technology in a small cohort of mice”. Larger animals are likely to progress in due course, including phase 1/2 human clinical trials, and the design of particular outcome measurements supportive of patient organisations. Finally, REVeRT may then expand further applications for other ophthalmic disorders, including Usher syndrome, congenital stationary night blindness and others.