Safe and effective liver-directed AAV-mediated homology-independent targeted integration in mouse models of inherited diseases.

Liver-directed adeno-associated viral (AAV) vector-mediated homology-independent targeted integration (AAV-HITI) by CRISPR-Cas9 at the highly transcribed albumin locus is under investigation to provide sustained transgene expression following neonatal treatment. We show that targeting the 3' end of the albumin locus results in productive integration in about 15% of mouse hepatocytes achieving therapeutic levels of systemic proteins in two mouse models of inherited diseases. We demonstrate that full-length HITI donor DNA is preferentially integrated upon nuclease cleavage and that, despite partial AAV genome integrations in the target locus, no gross chromosomal rearrangements or insertions/deletions at off-target sites are found. In line with this, no evidence of hepatocellular carcinoma is observed within the 1-year follow-up. Finally, AAV-HITI is effective at vector doses considered safe if directly translated to humans providing therapeutic efficacy in the adult liver in addition to newborn. Overall, our data support the development of this liver-directed AAV-based knockin strategy.

A new age of precision gene therapy.

Gene therapy has become a clinical reality as market-approved advanced therapy medicinal products for the treatment of distinct monogenetic diseases and B-cell malignancies. This Therapeutic Review aims to explain how progress in genome editing technologies offers the possibility to expand both therapeutic options and the types of diseases that will become treatable. To frame these impressive advances in the context of modern medicine, we incorporate examples from human clinical trials into our discussion on how genome editing will complement currently available strategies in gene therapy, which still mainly rely on gene addition strategies. Furthermore, safety considerations and ethical implications, including the issue of accessibility, are addressed as these crucial parameters will define the impact that gene therapy in general and genome editing in particular will have on how we treat patients in the near future.

The Opportunities and Challenges of Gene Therapy for Treatment of Inherited Forms of Vision and Hearing Loss.

Inherited forms of blindness and deafness are highly prevalent and severe conditions that significantly impact the lives of millions of people worldwide. The lack of therapeutic options for these conditions poses a major socioeconomic burden. Over the last decades, gene therapy has proven to be a life changing treatment for hereditary and acquired forms of diseases, and extensive preclinical investigation in animal models of both retinal and inner ear disorders has highlighted promising translational opportunities for these disorders too. This led to dozens of clinical trials investigating the efficiency of gene therapy-based approaches, with some of the products for retinal conditions successfully reaching phase III of development or even market authorization. However, challenges remain for the use of gene therapy, which are related to both the features of the delivery vehicles currently available and characteristics of the retinal and inner ear disorders targeted. Therefore, further developments in gene therapy platforms' design, including exploitation of novel technologies such as genome editing, RNA-targeted therapies, and optogenetics, are actively ongoing, driving the field forward. In this study, we review the ongoing applications and achievements of gene therapy for treatment of inherited forms of blindness and deafness as well as the developments that are being pursued in the field to overcome the current limitations.

Inclusion of a degron reduces levelsof undesired inteins after AAV-mediated proteintrans-splicing in the retina.

Split intein-mediated protein trans-splicing expands AAV transfer capacity, thus overcoming the limited AAV cargo. However, non-mammalian inteins persist as trans-splicing by-products, and this could raise safety concerns for AAV intein clinical applications. In this study, we tested the ability of several degrons to selectively decrease levels of inteins after protein trans-splicing and found that a version of E. coli dihydrofolate reductase, which we have shortened to better fit into the AAV vector, is the most effective. We show that subretinal administration of AAV intein armed with this short degron is both safe and effective in a mouse model of Stargardt disease (STGD1), which is the most common form of inherited macular degeneration in humans. This supports the use of optimized AAV intein for gene therapy of both STGD1 and other conditions that require transfer of large genes.

Light-responsive microRNA miR-211 targets Ezrin to modulate lysosomal biogenesis and retinal cell clearance.

Vertebrate vision relies on the daily phagocytosis and lysosomal degradation of photoreceptor outer segments (POS) within the retinal pigment epithelium (RPE). However, how these events are controlled by light is largely unknown. Here, we show that the light-responsive miR-211 controls lysosomal biogenesis at the beginning of light-dark transitions in the RPE by targeting Ezrin, a cytoskeleton-associated protein essential for the regulation of calcium homeostasis. miR-211-mediated down-regulation of Ezrin leads to Ca2+ influx resulting in the activation of calcineurin, which in turn activates TFEB, the master regulator of lysosomal biogenesis. Light-mediated induction of lysosomal biogenesis and function is impaired in the RPE from miR-211-/- mice that show severely compromised vision. Pharmacological restoration of lysosomal biogenesis through Ezrin inhibition rescued the miR-211-/- phenotype, pointing to a new therapeutic target to counteract retinal degeneration associated with lysosomal dysfunction.

Can Adeno-Associated Viral Vectors Deliver Effectively Large Genes?

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 full-length 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.

Has retinal gene therapy come of age? From bench to bedside and back to bench.

Retinal gene therapy has advanced considerably in the past three decades. Initial efforts have been devoted to comprehensively explore and optimize the transduction abilities of gene delivery vectors, define the appropriate intraocular administration routes and obtain evidence of efficacy in animal models of inherited retinal diseases (IRDs). Successful translation in clinical trials of the initial promising proof-of-concept studies led to the important milestone of the first approved product for retinal gene therapy in both US and Europe. The unprecedented clinical development observed during the last decade in the field is however highlighting new challenges that will need to be overcome to bring gene therapy to fruition to a larger patient population within and beyond the realm of IRDs.

Intein-mediated protein trans-splicing expands adeno-associated virus transfer capacity in the retina.

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.

Adeno-Associated Viral Vectors as a Tool for Large Gene Delivery to the Retina.

Gene therapy using adeno-associated viral (AAV) vectors currently represents the most promising approach for the treatment of many inherited retinal diseases (IRDs), given AAV's ability to efficiently deliver therapeutic genes to both photoreceptors and retinal pigment epithelium, and their excellent safety and efficacy profiles in humans. However, one of the main obstacles to widespread AAV application is their limited packaging capacity, which precludes their use from the treatment of IRDs which are caused by mutations in genes whose coding sequence exceeds 5 kb. Therefore, in recent years, considerable effort has been made to identify strategies to increase the transfer capacity of AAV vectors. This review will discuss these new developed strategies, highlighting the advancements as well as the limitations that the field has still to overcome to finally expand the applicability of AAV vectors to IRDs due to mutations in large genes.

Seeing the Light after 25 Years of Retinal Gene Therapy.

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.

Dual AAV Vectors for Stargardt Disease.

Stargardt disease (STGD1), due to mutations in the large ABCA4 gene, is the most common inherited macular degeneration in humans. Attempts at developing gene therapy approaches for treatment of STGD1 are currently ongoing. Among all the vectors available for gene therapy of inherited retinal diseases, those based on adeno-associated viruses (AAV) are the most promising given the efficacy shown in various animal models and their excellent safety profile in humans, as confirmed in many ongoing clinical trials. However, one of the main obstacles for the use of AAV is their limited effective packaging capacity of about 5 kb. Taking advantage of the AAV genome's ability to concatemerize , others and we have recently developed dual AAV vectors to overcome this limit. We tested dual AAV vectors for ABCA4 delivery, and found that they transduce efficiently both mouse and pig photoreceptors , and rescue the Abca4-/- mouse retinal phenotype, indicating their potential for gene therapy of STGD1. This chapter details how we designed dual AAV vectors for the delivery of the ABCA4 gene and describes the techniques that can be explored to evaluate dual AAV transduction efficiency in vitro and in the retina, and their efficacy in the mouse model of STGD1.

Improved dual AAV vectors with reduced expression of truncated proteins are safe and effective in the retina of a mouse model of Stargardt disease.

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 of inherited retinal degenerations: prospects and challenges.

Because of its favorable anatomical and immunological characteristics, the eye has been at the forefront of translational gene therapy. Dozens of promising proofs of concept have been obtained in animal models of inherited retinal degenerations (IRDs), and some of them have been relayed to the clinic. The results from the first clinical trials for a congenital form of blindness have generated great interest and have demonstrated the safety and efficacy of intraocular administrations of viral vectors in humans. However, this progress has also generated new questions and posed challenges that need to be addressed to further expand the applicability of gene therapy in the eye, including safe delivery of viral vectors to the outer retina, treatment of dominant IRDs as well as of IRDs caused by mutations in large genes, and, finally, selection of the appropriate IRDs and patients to maximize the efficacy of gene transfer. This review summarizes the strategies that are currently being exploited to overcome these challenges and drive the clinical development of retinal gene therapy.

Gene Therapy of ABCA4-Associated Diseases.

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.

Vector platforms for gene therapy of inherited retinopathies.

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.

Effective delivery of large genes to the retina by dual AAV vectors.

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.