Retinal gene therapy with adeno-associated viral (AAV) vectors is safe and effective in humans. However, AAV's limited cargo capacity prevents its application to therapies of inherited retinal diseases due to mutations of genes over 5 kb, like Stargardt's disease (STGD) and Usher syndrome type IB (USH1B). Previous methods based on ‘forced’ packaging of large genes into AAV capsids may not be easily translated to the clinic due to the generation of genomes of heterogeneous size which raise safety concerns. Taking advantage of AAV's ability to concatemerize, we generated dual AAV vectors which reconstitute a large gene by either splicing (trans-splicing), homologous recombination (overlapping), or a combination of the two (hybrid). We found that dual trans-splicing and hybrid vectors transduce efficiently mouse and pig photoreceptors to levels that, albeit lower than those achieved with a single AAV, resulted in significant improvement of the retinal phenotype of mouse models of STGD and USH1B. Thus, dual AAV trans-splicing or hybrid vectors are an attractive strategy for gene therapy of retinal diseases that require delivery of large genes.

Inherited retinopathies (IR) are common untreatable blinding conditions. Most of them are inherited as monogenic disorders, due to mutations in genes expressed in retinal photoreceptors (PR) and in retinal pigment epithelium (RPE). The retina’s compatibility with gene transfer has made transduction of different retinal cell layers in small and large animal models via viral and non-viral vectors possible. The ongoing identification of novel viruses as well as modifications of existing ones based either on rational design or directed evolution have generated vector variants with improved transduction properties. Dozens of promising proofs of concept have been obtained in IR animal models with both viral and non-viral vectors, and some of them have been relayed to clinical trials. To date, recombinant vectors based on the adeno-associated virus (AAV) represent the most promising tool for retinal gene therapy, given their ability to efficiently deliver therapeutic genes to both PR and RPE and their excellent safety and efficacy profiles in humans. However, AAVs’ limited cargo capacity has prevented application of the viral vector to treatments requiring transfer of genes with a coding sequence larger than 5 kb. Vectors with larger capacity, i.e. nanoparticles, adenoviral and lentiviral vectors are being exploited for gene transfer to the retina in animal models and, more recently, in humans. This review focuses on the available platforms for retinal gene therapy to fight inherited blindness, highlights their main strengths and examines the efforts to overcome some of their limitations.

Retinal gene therapy with adeno-associated viral (AAV) vectors holds promises for treating inherited and noninherited diseases of the eye. Although clinical data suggest that retinal gene therapy is safe and effective, delivery of large genes is hindered by the limited AAV cargo capacity. Protein trans-splicing mediated by split inteins is used by single-cell organisms to reconstitute proteins. Here, we show that delivery of multiple AAV vectors each encoding one of the fragments of target proteins flanked by short split inteins results in protein trans-splicing and full-length protein reconstitution in the retina of mice and pigs and in human retinal organoids. The reconstitution of large therapeutic proteins using this approach improved the phenotype of two mouse models of inherited retinal diseases. Our data support the use of split intein–mediated protein trans-splicing in combination with AAV subretinal delivery for gene therapy of inherited blindness due to mutations in large genes.

The retina has been at the forefront of translational gene therapy. Proof-of-concept that gene therapy could restore vision in a large animal led to the initiation of the first successful clinical trials and, in turn, to the recent approval of the first gene therapy product for an ocular disease. As dozens of clinical trials of retinal gene therapy have begun, new challenges are identified, which include delivery of large genes, counteracting gain-of-function mutations, and safe and effective gene transfer to diseased retinas. Advancements in vector design, improvements of delivery routes, and selection of optimal timing for intervention will contribute to extend the initial success of retinal gene therapy to an increasing number of inherited blinding conditions.

Gene therapy with adeno-associated viral (AAV) vectors is limited by AAV cargo capacity that prevents their application to the inherited retinal diseases (IRDs), such as Stargardt disease (STGD) or Usher syndrome type IB (USH1B), which are due to mutations in genes larger than 5 kb. Trans-splicing or hybrid dual AAV vectors have been successfully exploited to reconstitute large gene expression in the mouse retina. Here, we tested them in the large cone-enriched pig retina that closely mimics the human retina. We found that dual AAV trans-splicing and hybrid vectors transduce pig photoreceptors, the major cell targets for treatment of IRDs, to levels that were about two- to threefold lower than those obtained with a single AAV vector of normal size. This efficiency is significantly higher than that in mice, and is potentially due to the high levels of dual AAV co-transduction we observe in pigs. We also show that subretinal delivery in pigs of dual AAV trans-splicing and hybrid vectors successfully reconstitute, albeit at variable levels, the expression of the large genes ABCA4 and MYO7A mutated in STGD and USH1B, respectively. Our data support the potential of dual AAV vectors for large gene reconstitution in the cone-enriched pig retina that is a relevant preclinical model.

Stargardt disease (STGD1) due to mutations in the large ABCA4 gene is the most common inherited macular degeneration in humans. We have shown that dual adeno-associated viral (AAV) vectors effectively transfer ABCA4 to the retina of Abca4−/− mice. However, they express both lower levels of transgene compared with a single AAV and truncated proteins. To increase productive dual AAV concatemerization, which would overcome these limitations, we have explored the use of either various regions of homology or heterologous inverted terminal repeats (ITR). In addition, we tested the ability of various degradation signals to decrease the expression of truncated proteins. We found the highest levels of transgene expression using regions of homology based on either alkaline phosphatase or the F1 phage (AK). The use of heterologous ITR does not decrease the levels of truncated proteins relative to full-length ABCA4 and impairs AAV vector production. Conversely, the inclusion of the CL1 degradation signal results in the selective degradation of truncated proteins from the 5′-half without affecting full-length protein production. Therefore, we developed dual AAV hybrid ABCA4 vectors including homologous ITR2, the photoreceptor-specific G protein-coupled receptor kinase 1 promoter, the AK region of homology and the CL1 degradation signal. We show that upon subretinal administration these vectors are both safe in pigs and effective in Abca4−/− mice. Our data support the use of improved dual AAV vectors for gene therapy of STGD1.

Gene therapy with adeno-associated viral (AAV) vectors has reached the clinical stage for many inherited and acquired diseases. However, due to a cargo capacity limited to <5 kb, AAV-mediated treatment of diseases that require transfer of larger genes still appears elusive. This is a major drawback of a platform that has otherwise been repeatedly found to be safe and effective. Thus, great efforts have been directed toward the identification of strategies to overcome this limitation. Among the most studied approaches is the use of dual vectors, in which a transgene is split across two separate AAV vectors. Mechanisms acting at either the DNA, pre-mRNA, or protein levels have been explored to restore fulllength transgene expression in infected cells. Here, we will review them as well as additional strategies developed to deliver large genes with AAV. We discuss the pros and cons of these strategies and the aspects that still need to be addressed.

The ATP-binding cassette (ABC) transporter gene, ABCA4 (ABCR), was characterized in 1997 as the causal gene for autosomal recessive Stargardt disease (STGD1). Shortly thereafter several other phenotypes were associated with mutations in ABCA4, which now have collectively emerged as the most frequent cause of retinal degeneration phenotypes of Mendelian inheritance. ABCA4 functions as an important transporter (or "flippase") of vitamin A derivatives in the visual cycle. Several ways to alleviate the effects of the defective ABCA4 protein, which cause accumulation of 11-cis and all-trans-retinal in photoreceptors and lipofuscin in the retinal pigment epithelium, have been proposed. Although ABCA4 has proven to be a difficult research target, substantial progress through genetic, functional, and translational studies has allowed major advances in therapeutic applications for ABCA4-associated pathology, which should be available to patients in the (near) future. Here, we summarize the status of the gene therapy-based treatment options of ABCA4-associated diseases.