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The degeneration of light-detecting rod and cone photoreceptors in the human retina leads to severe visual impairment and ultimately legal blindness in millions of people worldwide. Multiple therapeutic options at different stages of degeneration are being explored but the majority of ongoing clinical trials involve adeno-associated viral (AAV) vector-based gene supplementation strategies for select forms of inherited retinal disease. Over 300 genes are associated with inherited retinal degenerations and only a small proportion of these will be suitable for gene replacement therapy. However, while the origins of disease may vary, there are considerable similarities in the physiological changes that occur in the retina. When early therapeutic intervention is not possible and patients suffer loss of photoreceptor cells but maintain remaining layers of cells in the neural retina, there is an opportunity for a universal gene therapy approach that can be applied regardless of the genetic origin of disease. Optogenetic therapy offers such a strategy by aiming to restore vision though the provision of light-sensitive molecules to surviving cell types of the retina that enable light perception through the residual neurons. Here we review the recent progress in attempts to restore visual function to the degenerate retina using optogenetic therapy. We focus on multiple pre-clinical models used in optogenetic strategies, discuss their strengths and limitations, and highlight considerations including vector and transgene designs that have advanced the field into two ongoing clinical trials.

Rhodopsin (RHO) gene mutations are a common cause of autosomal dominant retinitis pigmentosa (ADRP). The need to suppress toxic protein expression together with mutational heterogeneity pose challenges for treatment development. Mirtrons are atypical RNA interference effectors that are spliced from transcripts as short introns. Here, we develop a novel mirtron-based knockdown/replacement gene therapy for the mutation-independent treatment of RHO-related ADRP, and demonstrate efficacy in a relevant mammalian model. Splicing and potency of rhodopsin-targeting candidate mirtrons are initially determined, and a mirtron-resistant codon-modified version of the rhodopsin coding sequence is validated in vitro. These elements are then combined within a single adeno-associated virus (AAV) and delivered subretinally in a Rho P23H knock-in mouse model of ADRP. This results in significant mouse-to-human rhodopsin RNA replacement and is associated with a slowing of retinal degeneration. This provides proof of principle that synthetic mirtrons delivered by AAV are capable of reducing disease severity in vivo. Rhodopsin-related dominant retinitis pigmentosa is a degenerative disease of the retina of the eye for which there is no current treatment. In this study, the authors use a novel form of RNA-interference- artificial mirtrons- to slow retinal degeneration in a mouse model of the disease.

Non-viral gene therapy has the potential to overcome several shortcomings in viral vector-based therapeutics. Methods of in vivo plasmid delivery have developed over recent years to increase the efficiency of non-viral gene transfer, yet further improvements still need to be made to improve their translational capacity. Gene therapy advances for inherited retinal disease have been particularly prominent over the recent decade but overcoming physical and physiological barriers present in the eye remains a key obstacle in the field of non-viral ocular drug delivery. Minicircles are circular double-stranded DNA vectors that contain expression cassettes devoid of bacterial DNA, thereby limiting the risks of innate immune responses induced by such elements. To date, they have not been extensively used in pre-clinical studies yet remain a viable vector option for the treatment of inherited retinal disease. Here, we explore the potential of minicircle DNA delivery to the neural retina as a gene therapy approach. We consider the advantages of minicircles as gene therapy vectors as well as review the challenges involved in optimising their delivery to the neural retina.

Photoreceptor degeneration due to retinitis pigmentosa (RP) is a primary cause of inherited retinal blindness. Photoreceptor cell-replacement may hold the potential for repair in a completely degenerate retina by reinstating light sensitive cells to form connections that relay information to downstream retinal layers. This study assessed the therapeutic potential of photoreceptor progenitors derived from human embryonic and induced pluripotent stem cells (ESCs and iPSCs) using a protocol that is suitable for future clinical trials. ESCs and iPSCs were cultured in four specific stages under defined conditions, resulting in generation of a near-homogeneous population of photoreceptor-like progenitors. Following transplantation into mice with end-stage retinal degeneration, these cells differentiated into photoreceptors and formed a cell layer connected with host retinal neurons. Visual function was partially restored in treated animals, as evidenced by two visual behavioral tests. Furthermore, the magnitude of functional improvement was positively correlated with the number of engrafted cells. Similar efficacy was observed using either ESCs or iPSCs as source material. These data validate the potential of human pluripotent stem cells for photoreceptor replacement therapies aimed at photoreceptor regeneration in retinal disease.

Uveitis is characterised by breakdown of the blood-retinal barrier (BRB), allowing infiltration of immune cells that mediate intraocular inflammation, which can lead to irreversible damage of the neuroretina and the loss of sight. Treatment of uveitis relies heavily on corticosteroids and systemic immunosuppression due to limited understanding of disease pathogenesis. We performed single-cell RNA-sequencing of retinas, as well as bulk RNA-sequencing of retinal pigment epithelial (RPE) cells from mice with experimental autoimmune uveitis (EAU) versus healthy control. This revealed that the Th1/Th17-driven disease induced strong gene expression changes in response to inflammation in rods, cones, Müller glia and RPE. In particular, Müller glia and RPE cells were found to upregulate expression of chemokines, complement factors, leukocyte adhesion molecules and MHC class II, thus highlighting their contributions to immune cell recruitment and antigen presentation at the inner and outer BRB, respectively. Additionally, ligand-receptor interaction analysis with CellPhoneDB revealed key interactions between Müller glia and T cell / natural killer cell subsets via chemokines, galectin-9 to P4HB/TIM-3, PD-L1 to PD-1, and nectin-2/3 to TIGIT signalling axes. Our findings elucidate mechanisms contributing to breakdown of retinal immune privilege during uveitis and identify novel targets for therapeutic interventions.

RNA editing holds great promise for the therapeutic correction of pathogenic, single nucleotide variants (SNV) in the human transcriptome since it does not risk creating permanent off-targets edits in the genome and has the potential for innovative delivery options. Adenine deaminases acting on RNA (ADAR) enzymes catalyse the most widespread form of posttranscriptional RNA editing in humans and their ability to hydrolytically deaminate adenosine to inosine in double stranded RNA (dsRNA) has been harnessed to change pathogenic single nucleotide variants (SNVs) in the human genome on a transcriptional level. Until now, the most promising target editing rates have been achieved by exogenous delivery of the catalytically active ADAR deaminase domain (ADAR DD ) fused to an RNA binding protein. While it has been shown that endogenous ADARs can be recruited to a defined target site with the sole help of an ADAR-recruiting guide RNA, thus freeing up packaging space, decreasing the chance of an immune response against a foreign protein, and decreasing transcriptome-wide off-target effects, this approach has been limited by a low editing efficiency. Through the recent development of novel circular ADAR-recruiting guide RNAs as well as the optimisation of ADAR-recruiting antisense oligonucleotides, RNA editing with endogenous ADAR is now showing promising target editing efficiency in vitro and in vivo . A target editing efficiency comparable to RNA editing with exogenous ADAR was shown both in wild-type and disease mouse models as well as in wild-type non-human primates (NHP) immediately following and up to 6 weeks after application. With these encouraging results, RNA editing with endogenous ADAR has the potential to present an attractive option for the treatment of inherited retinal diseases (IRDs), a field where gene replacement therapy has been established as safe and efficacious, but where an unmet need still exists for genes that exceed the packaging capacity of an adeno associated virus (AAV) or are expressed in more than one retinal isoform. This review aims to give an overview of the recent developments in the field of RNA editing with endogenous ADAR and assess its applicability for the field of treatment of IRD.

Optogenetic strategies to restore vision in patients who are blind from end-stage retinal degenerations aim to render remaining retinal cells light sensitive once photoreceptors are lost. Here, we assessed long-term functional outcomes following subretinal delivery of the human melanopsin gene (OPN4) in the rd1 mouse model of retinal degeneration using an adeno-associated viral vector. Ectopic expression of OPN4 using a ubiquitous promoter resulted in cellular depolarization and ganglion cell action potential firing. Restoration of the pupil light reflex, behavioral light avoidance, and the ability to perform a task requiring basic image recognition were restored up to 13 mo following injection. These data suggest that melanopsin gene therapy via a subretinal route may be a viable and stable therapeutic option for the treatment of end-stage retinal degeneration in humans.

Despite being the most prevalent cause of inherited blindness in children, Stargardt disease is yet to achieve the same clinical trial success as has been achieved for other inherited retinal diseases. With an early age of onset and continual progression of disease over the life course of an individual, Stargardt disease appears to lend itself to therapeutic intervention. However, the aetiology provides issues not encountered with the likes of choroideremia and X-linked retinitis pigmentosa and this has led to a spectrum of treatment strategies that approach the problem from different aspects. These include therapeutics ranging from small molecules and anti-sense oligonucleotides to viral gene supplementation and cell replacement. The advancing development of CRISPR-based molecular tools is also likely to contribute to future therapies by way of genome editing. In this we review, we consider the most recent pre-clinical and clinical trial data relating to the different strategies being applied to the problem of generating a treatment for the large cohort of Stargardt disease patients worldwide.

X-linked retinitis pigmentosa (XLRP) is generally a severe form of retinitis pigmentosa, a neurodegenerative, blinding disorder of the retina. 70% of XLRP cases are due to mutations in the retina-specific isoform of the gene encoding retinitis pigmentosa GTPase regulator (RPGRORF15). Despite successful RPGRORF15 gene replacement with adeno-associated viral (AAV) vectors being established in a number of animal models of XLRP, progression to human trials has not yet been possible. The inherent sequence instability in the purine-rich region of RPGRORF15 (which contains highly repetitive nucleotide sequences) leads to unpredictable recombination errors during viral vector cloning. While deleted RPGR may show some efficacy in animal models, which have milder disease, the therapeutic effect of a mutated RPGR variant in patients with XLRP cannot be predicted. Here, we describe an optimized gene replacement therapy for human XLRP disease using an AAV8 vector that reliably and consistently produces the full-length correct RPGR protein. The glutamylation pattern in the RPGR protein derived from the codon-optimized sequence is indistinguishable from the wild-type variant, implying that codon optimization does not significantly alter post-translational modification. The codon-optimized sequence has superior stability and expression levels in vitro. Significantly, when delivered by AAV8 vector and driven by the rhodopsin kinase promoter, the codon-optimized RPGR rescues the disease phenotype in two relevant animal models (Rpgr−/y and C57BL/6JRd9/Boc) and shows good safety in C57BL6/J wild-type mice. This work provides the basis for clinical trial development to treat patients with XLRP caused by RPGR mutations. X-linked retinitis pigmentosa caused by mutations in RPGR is a frequent cause of retinal degeneration and blindness without available treatment. Fischer et al. demonstrate safety and efficacy of gene supplementation in relevant animal models using a codon-optimized transgene, thereby resolving the problem of sequence instability of wild-type RPGR.

Many genetic diseases and undesirable traits are due to base-pair alterations in genomic DNA. Base-editing, the newest evolution of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas-based technologies, can directly install point-mutations in cellular DNA without inducing a double-strand DNA break (DSB). Two classes of DNA base-editors have been described thus far, cytosine base-editors (CBEs) and adenine base-editors (ABEs). Recently, prime-editing (PE) has further expanded the CRISPR-base-edit toolkit to all twelve possible transition and transversion mutations, as well as small insertion or deletion mutations. Safe and efficient delivery of editing systems to target cells is one of the most paramount and challenging components for the therapeutic success of BEs. Due to its broad tropism, well-studied serotypes, and reduced immunogenicity, adeno-associated vector (AAV) has emerged as the leading platform for viral delivery of genome editing agents, including DNA-base-editors. In this review, we describe the development of various base-editors, assess their technical advantages and limitations, and discuss their therapeutic potential to treat debilitating human diseases.