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Gene Therapy for Inherited Retinal Disease: Original Articles

Retinal Gene Therapy for Usher Syndrome: Current Developments, Challenges, and Perspectives

Dinculescu, Astra PhD; Link, Brian A. PhD; Saperstein, David A. MD

Author Information
International Ophthalmology Clinics: Fall 2021 - Volume 61 - Issue 4 - p 109-124
doi: 10.1097/IIO.0000000000000378
  • Open


Usher syndrome (USH) represents a group of genetically heterogenous autosomal recessive disorders, characterized by combined vision and sensorineural hearing loss, and in some cases vestibular dysfunction.1–3 It primarily affects the light-sensitive photoreceptor cells in the retina and the auditory hair cells in the cochlea. Vision loss in all cases is progressive, and manifests as retinitis pigmentosa (RP), characterized by the gradual degeneration of the photoreceptor cells.4 Peripheral rod function is lost first, leading to night blindness and constricted visual fields, followed by the death of cones and severe visual impairment. There are currently no drugs or biological therapies proven to be effective in treating USH syndrome. Cochlear implantation, which bypasses the damaged hair cells and stimulates the primary auditory neurons directly, is an effective approach to alleviate the hearing impairment in patients. However, there is no remedy for the progressive loss of the photoreceptor neurons in the retina, and thus a critical unmet need exists to develop therapeutic strategies to prevent blindness in USH syndrome. The generation of animal models that faithfully mimic the human USH disorder, the timing of gene therapy interventions, the identification of correct target cells and patient selection are key factors in successfully reaching this goal.

USH syndrome was named after Dr Charles Howard Usher, a Scottish ophthalmologist who described 69 patients with the disease in 1914.5 As is commonly the case in medicine, the disease was first described much earlier (1858) in brothers suffering from blindness and deafness by von Gräfe6 and was further characterized by his student, Liebreich,7 3 years later in a larger population of patients. From its discovery until recently, USH syndrome and its subgroups were delineated by clinical characteristics.8 Now the disease is recognized by symptomology and classified by genotyping. To date, there are at least 10 causative genes that are associated with USH syndrome, summarized in previous reviews.1,2,9 In general, based on its diverse clinical symptoms, particularly the onset and severity of the sensorineural hearing impairment and the presence of vestibular dysfunction, USH syndrome is grouped into 3 clinical subtypes. Type I disease is associated with severe-to-profound prelingual hearing loss, vestibular abnormalities and prepubescent onset of RP symptoms.10,11 USH1 results from mutations in MYO7A (USH1B), HARMONIN (USH1C), CDH23 (USH1D), PCDH15 (USH1F), SANS (USH1G), and CIB2 (USH1J). Patients with type II disease display moderate-to-severe congenital hearing loss without vestibular dysfunction, and account for >50% of all USH cases. Their RP symptoms generally begin in the second decade.12 USH2 is caused by mutations in USH2A (USH2A), ADGRV1 (USH2C), and WHRN (USH2D). Patients with type III disease caused by mutations in CLRN1 (USH3A) gene have postlingual progressive hearing loss, variable vestibular dysfunction, and variable onset of RP that rapidly progresses to legal blindness by the fourth decade of life.13–17 Mutations in the HARS gene (Histidyl-tRNA synthetase) are associated with the ultra-rare form USH3B.18,19 Other genes have been described in cases of atypical USH, including: CEP250 and CEP78, encoding members of the CEP family of centrosome-associated proteins; ESPN, encoding the F-actin cross-linker espin; ARSG, encoding the arylsulfatase G enzyme.20

Gene Therapy for USH Syndrome: Current Challenges

The success of gene therapy for Leber Congenital Amaurosis (LCA2) due to defects in the RPE65 gene and subsequent FDA and EMA approval of Luxturna (voretigene neparvovec) have sparked hope that many other autosomal recessive retinal (and neurological) diseases may also be treated using a vector-based gene delivery approach.21,22 USH syndrome was no exception and several laboratories embarked on USH gene transfer studies in animal models. Conventional recombinant adeno-associated viral vectors (AAVs) contain a single-stranded DNA expression cassette, which includes a cell-specific or ubiquitous promoter, the transgene and polyadenylation signal, flanked by palindromic inverted terminal repeats at both ends. In contrast to other gene therapy systems, AAV vectors have low immunogenicity (especially when given subretinally), display long-lasting expression in postmitotic cells due to their ability to primarily persist as episomes, and efficiently transduce both photoreceptors and the retinal pigmented epithelium (RPE), where most RP-associated genes are expressed.23 Overall, AAV vectors hold tremendous potential to prevent retinal degeneration in USH syndrome. However, there are numerous challenges in the field, which become apparent by acknowledging the critical disease-specific parameters that account for the success of AAV-based gene augmentation therapy in RPE65 LCA. These include the availability of small and large animal models of RPE65-deficiency displaying a robust phenotype, a slow degenerative component of the disease accompanying a marked retinal dysfunction, a large window for treatment, and the lack of detrimental effects following long-term AAV-mediated transgene overexpression in RPE.24 In contrast to ocular diseases in USH syndrome, the molecular basis of retinal dysfunction in LCA2 is well understood. Moreover, the lack of RPE65 causes a substantial loss of visual function in murine and canine animal models, as demonstrated by the significantly reduced or nonrecordable electroretinograms.25–27 The marked loss of vision in animal models, which contrasts sharply with the remarkable preservation of retinal architecture, has allowed testing of long-term gene therapy effects.28–30 The RPE65 protein is a 65 kDa retinoid isomerohydrolase, which plays an essential role in the retinoid visual cycle by converting all-trans-retinyl ester to 11-cis-retinol in RPE cells.31–33 The 11-cis retinal chromophore deficiency in LCA2 photoreceptor outer segments can be corrected by restoring RPE65 enzyme to RPE, either by using a cell-specific human RPE65 promoter or a modified version of the ubiquitous chicken-beta actin promoter. Furthermore, one RPE cells interdigitates with 20 to 40 photoreceptor cells at the apical interface, amplifying the effects of gene therapy while minimizing the potential downside of nonuniform AAV-mediated transgene expression in neighboring RPE cells. Subretinal injection of an AAV2 vector delivering the canine wild-type RPE65 cDNA in dog models has produced long-term beneficial effects on visual function and protection from degeneration especially when treatment was initiated early.29,30,34,35 Ultimately, the successful restoration of vision in a large animal model paved the way towards an AAV-based gene therapy for LCA2 patients.36–40

Unlike the case of RPE65-associated disease, the majority of the genetically engineered mouse models of USH syndrome do not exhibit a visual phenotype, although they all display hearing loss.41,42 Some of these models were reported to display only a progressive retinal dysfunction with aging.42 It has been suggested that the lack of a retinal phenotype in mouse models could result from a distinct species-specific cell pattern of gene expression or to morphologic differences between human and mouse photoreceptors, for example, mice lack the well-developed, actin-filled calyceal processes, the subcellular structures extending from the inner segments in frogs, pigs, zebrafish, and primates.43,44 The absence of a robust ocular phenotype in animal models has severely limited the ability to perform therapeutic, preclinical proof-of-concept testing for vision loss in USH syndrome. However, gene replacement, gene editing, and small molecule therapy have been used to preserve hearing in many USH animal models with positive results.9,45–53 There are several recent, in-depth reviews of these therapies.9,54,55 These data, combined with the progressive nature of the vision loss due to USH syndrome, have motivated a few companies to empirically study treatments on human subjects. One example is the lentiviral-mediated MYO7A gene delivery in USH1B.56,57 This approach was shown to correct the melanosome mislocalization and opsin accumulation at the photoreceptor connecting cilium in the Myo7a-deficient shaker1 mouse model of USH1B.57 Conventional AAV has a small packaging capacity compared with lentiviral vector (4.7 vs. 8.5 kb). MYO7A cDNA, which is expressed in photoreceptors and the RPE cells, is too large (~6.7 kb) to be packaged in AAV vectors.58 Therefore, an equine infectious anemia virus-based lentiviral vector (equine infectious anemia virus-CMV-MYO7A, UshStat) was subretinally delivered to USH1B patients with the goal to prevent or slow down the RP progression ( identifier: NCT01505062). Long-term safety evaluation of this treatment in patients who received UshStat is currently being performed in a clinical trial (NCT02065011, results pending). Another clinical trial (UshTher) is currently underway, which uses a dual AAV vector approach to express MYO7A in the retina of patients following subretinal delivery ( Dual AAV vectors take advantage of the ability of the 2 distinct AAV genomes containing the 5′ and 3′ half of MYO7A transgene cDNA to undergo intermolecular concatamerization, followed by homologous recombination to reconstitute the whole large gene expression cassette.59,60 To date, there has been no clinically positive data reported from either one of these trials.

The disease associated with much larger genes, for example, USH2A (cDNA~15.6 kb), may benefit from genome editing approaches, such as CRISPR-Cas9-sgRNAs, and those that promote exon skipping to restore functional protein restoration.61–66 A splicing-modulating antisense oligonucleotide approach designed to cause in-frame skipping of the mutated exon 13 of the USH2A disease-associated transcripts results in a shortened but functional usherin protein, which localizes correctly in photoreceptors and hair cells.66–68 Interestingly, recent studies have shown that zebrafish models of USH2A disease display early-onset retinal dysfunction, indicating they may represent useful tools to investigate the pathophysiology underlying USH syndrome and test therapeutic strategies.69,70 Morpholino antisense oligonucleotides were shown to mediate ush2a exon 13 skipping and rescue the visual dysfunction in a zebrafish USH2A model.66 This mutation-specific therapeutic strategy is being evaluated in a clinical trial using an antisense oligonucleotide (QR-421a) administered through intravitreal injections, with early evidence of efficacy reported at the 3-month interim analysis (phase 1/2 Stellar trial of QR-421a, ProQR Therapeutics, ID: NCT03780257).

Critical for the success of future therapeutic approaches is the generation and characterization of novel animal models that mimic the human ocular USH disease. These models will not only serve as preclinical platforms to test potential therapies, including viral-based gene delivery strategies, but can also be used to gain insight into the molecular mechanisms underlying retinal dysfunction and degeneration in USH syndrome. Interestingly, a pig model of USH1C lacking harmonin, USH1C (R31X), was recently reported to mimic the human disease, displaying combined deafness, changes in photoreceptor architecture, and significantly reduced visual function.71

USH syndrome has an estimated prevalence ranging from 4 to 17 cases per 100,000 individuals worldwide.72,73 Approximately 98% of USH syndrome patients belong to either type 1 or type 2 subgroups of disease. In contrast, USH3 is relatively rare, accounting for ~2% of all cases.74 However, it is the most prevalent form of USH disease in the Finnish population, where it represents an estimated 40% of all USH cases, and it also segregates to a high degree in patients of Ashkenazi-Jewish descent.16,17,74 Through funding from the Usher III initiative, a team of researchers was brought together in 2007 to uncover the roles of CLRN1 in sensory organs and the pathology that ensues when the gene is defective. Several therapeutic approaches were developed as a result of this sustained effort, with the ultimate goal of treating USH3A patients. An offshoot of this effort also resulted in several NIH-funded grants specifically directed towards understanding the complex pathophysiology of USH3 in the retina and cochlea. The rest of this review will highlight important findings of this research, and discuss strategies for developing a potentially effective gene therapy approach for USH syndrome.

The USH Protein Network

The lack of therapeutic approaches to prevent vision loss in USH patients has motivated a considerable amount of research on the pathophysiology of disease. Fluorescence colocalization studies on tissue sections from different species and reciprocal pull-down assays suggest that all proteins encoded by USH genes interact with each other and organize into large complexes within specific subcellular regions in photoreceptors and hair cells.75 They form highly dynamic networks, which consist of proteins from different classes with critical roles in transport, adhesion, and signaling processes in both sensory cell types. USH proteins include the actin-based motor protein myosin VIIa (MYO7A, USH1B); the cell-adhesion transmembrane proteins cadherin 23 (CDH23, USH1D), protocadherin 15 (PCDH15, USH1F), and usherin (USH2A); the very-large G protein-coupled receptor-1 VLGR1 (ADGRV1, GPR98, USH2C); the calcium and integrin binding protein 2 (CIB2, USH1J); the PDZ-domain scaffolding proteins harmonin (USH1C) and whirlin (WHRN, USH2D); the ankyrin and SAM-domain containing scaffolding protein SANS (USH1G); the tetraspanin-like protein clarin-1 (CLRN1, USH3A). In the retina, USH proteins were found to concentrate in specialized regions of photoreceptor cells, the connecting cilium, calyceal processes, and periciliary membrane.2 Some of the USH proteins were also detected at ribbon synapses in both photoreceptors and hair cells, which enable a rapid and sustained release of neurotransmitters necessary for the high frequency transmission of signals in sensory cells.76

The auditory system has often served as a mirror to gain insight into potential functions of USH proteins in the retina. In the cochlea, USH proteins were detected in the hair cell bundle, a mechanosensory antenna containing numerous F-actin rich stereocilia projecting from the apical surface of these auditory cells. As an elegant example, PCDH15 and CDH23 form both transient and permanent extracellular filamentous links between neighboring stereocilia, while harmonin, Sans, and myosin VIIa anchor these links to the intracellular actin core.77–80 In adult stages, PCDH15 and CDH23 form tip links, suggested to act similar to gating springs and open the sensory mechanoelectrical transduction ion channels in response to mechanical stimuli provided by sound waves.81,82 In the retina, USH1 proteins were detected at the fragile actin-rich calyceal processes in macaque, zebrafish, and humans, and may play a role in their development and maintenance.43,83 Similar to the interstereociliary links found in the hair bundle, USH1 proteins may form and stabilize membrane-membrane connection sites linking the calyceal processes to photoreceptor outer segments, allowing them to withstand daily mechanical stress.43 Consistent with this possibility, USH proteins organize and stabilize interactions between microvilli of absorptive intestinal cells.84,85

In contrast to USH1, the USH2 proteins usherin, VLGR1, and whirlin are mainly located in the periciliary ridge membrane complex region surrounding the photoreceptor connecting cilium, where they provide structural support and may also participate in vesicle docking with the plasma membrane during protein translocation to the outer segment.63,86 Pathogenic mutations leading to dysfunction or absence of a particular USH protein were found to alter the localization of other members in the network, a property which has been exploited to evaluate the efficacy of gene therapy experiments in animal models.87 Considering their subcellular localization to specific regions critical for transport and structural maintenance, careful AAV vector dosing and choice of regulatable promoters in gene therapy studies will be needed to ensure that long-term vector-mediated USH proteins expression mimics endogenous levels and does not lead to detrimental effects on photoreceptors structure and function.88 The use of novel penetrating AAV capsids with higher transduction efficiency may enable photoreceptor targeting following a surgically safer intravitreal approach, reducing the concentration of vector reaching the outer retina, and avoiding potential overexpression effects.89,90

CLRN1: an “Invisible” Tetraspanin Protein of Unknown Biological Function

A major challenge in the USH field lies in the difficulty of reliably detecting the USH proteins in the retina, due to limited epitope availability, a consequence of USH protein complexes and their low levels of expression. This aspect, combined with the lack of thoroughly validated antibody reagents and proper negative controls in certain cases, has generated conflicting information regarding the cellular expression pattern of some of the USH proteins in the retina. This is especially true for CLRN1, a tetraspanin-like glycoprotein whose biological function is currently unknown.91 The main CLRN1 isoform encodes a 232 amino acid protein that contains a single glycosylation site at asparagine 48 (N48) in the first extracellular loop and a potential PDZ binding motif within its C-terminal end.91,92 In vitro experiments using a hemagglutinin (HA)-epitope tagged CLRN1 have shown that following its overexpression in HEK cells, the protein concentrates at specific regions within plasma membrane and forms CLRN1-enriched microdomains, recruiting diverse proteins involved in cell-adhesion, focal adhesion, and tight junctions, with an essential role in organizing the actin cytoskeleton.93

In the cochlea, CLRN1 has been suggested to interact with USH1 proteins harmonin and PCDH15.47,94Clrn1 knockout and the N48K knock-in mouse models of USH3 do not develop a retinal degeneration phenotype, but display an early-onset hearing loss and disorganized F-actin-rich stereocilia, indicating that one function of CLRN1 in the cochlea is to maintain the structural integrity of the hair bundle.95–97 A zebrafish model further established that CLRN1 is as an essential hair bundle protein.98 Other studies reported that CLRN1 absence also causes synaptic defects in cochlea hair cells.47,94 Three recent studies have shown that gene therapy can successfully prevent hearing loss in different mouse models of USH3 following early postnatal AAV-mediated Clrn1 cochlear delivery, when the sensory hair cell structure is preserved.47–49 These proof of principle studies raise hope that AAV-mediated CLRN1 expression to the correct retinal target cells will also be successful, providing that therapy is initiated before significant structural damage to photoreceptor cells occurs.

Studies aimed to define the cellular expression of endogenous CLRN1 in the eye detected it either in the inner or outer retinal cells, or both, depending on the method used (in situ hybridization for mRNA or immunostaining for protein).97,99–101 Localization was further confounded by differences in RNA hybridization probes or antibodies used, variability in sample processing conditions, as well as the particular species analyzed. In one study, Clrn1 transcripts were transiently detected during postnatal development in the inner retina by in situ hybridization (ISH), in a pattern consistent with progenitor cells and Müller glia expression.97 By using rodless aged rd1 mice and laser capture microdissection, that study further showed that in adult mouse retinas Clrn1 mRNA was confined to the inner nuclear layer, detectable only by RT-PCR. In the same year, another group reported that CLRN1 protein was expressed in photoreceptor synapses and connecting cilium regions following immunostaining of the mouse retina with a custom made anti-CLRN1 antibody.101 In zebrafish, CLRN1 was detected in both the inner and outer retina.99 However, several studies concluded that endogenous CLRN1 protein remains below the levels of detection of immunostaining.47,49,97,100 Recently, we have generated and characterized a novel N-terminus epitope-tagged CLRN1 knock-in mouse to facilitate the detection of endogenous CLRN1 protein expression in retinal sections with a high affinity anti-HA antibody.100 We noted a similar background labeling pattern in both HA-tagged CLRN1 knock-in and the C57BL/6J controls. Importantly, the HA-tagged CLRN1 in this mouse was only detectable by western blot analysis of retinal homogenates, and was expressed continuously during postnatal development and adulthood.100 The HA-tagged CLRN1 protein in retinal sections could only be successfully detected following its viral-mediated overexpression using a subretinal or intravitreal delivery approach.100,102

The availability of single-cell RNA sequencing (scRNAseq) technology has provided an unprecedented opportunity to firmly map the localization of CLRN1 and other USH gene transcripts in the retina in multiple species, providing a complementary tool to immunodetection.103,104 In our recently published study, by using scRNAseq analysis of large data sets, we bypassed the challenges of immunostaining experiments, and convincingly demonstrated that CLRN1 transcripts in adult retina are present in Müller glia in human, mouse and non-human primates.100 Müller glia enrichment of CLRN1 transcripts in the retina has been corroborated in human samples by other scRNAseq studies.105–108 Interestingly, scRNAseq data sets have provided evidence that HARMONIN (USH1C), CDH23 (USH1D), and SANS (USH1G) transcripts are also present Müller glia, supporting the need to consider the involvement of multiple cell types in USH disease.108 Furthermore, by using the highly sensitive RNAscope ISH assay we detected CLRN1 transcripts in the inner retina and not photoreceptors, both in the adult mouse and human tissue, a result consistent with the scRNAseq data.100 No signal was detected when the ISH Clrn1 target probes were used on Clrn1 knockout mouse sections, confirming the high specificity of the assay.100 We have recently examined CLRN1 mRNA expression in the pig retina using a similar RNAscope ISH chromogenic assay. We determined that CLRN1 transcripts are abundantly expressed early in postnatal development, before the photoreceptor outer segments are fully developed, concentrating within the middle part of the inner nuclear layer of piglet retinas (Fig. 1). Taken together, these experiments provide solid evidence that CLRN1 transcripts are expressed in the inner retina in multiple species, and highlight the complexities inherent in understanding the molecular pathophysiology of vision loss in USH syndrome and potentially, a novel biology associated with retinal degeneration caused by Müller glia dysfunction. However, these studies do not completely rule out the possibility that CLRN1 might also be expressed in photoreceptor cells at very low levels, undetectable with our current technologies.

Figure 1:
Detection of CLRN1 transcripts in piglet retina at postnatal day P2 using the RNAscope ISH assay (red punctate staining). GCL indicates ganglion cell layer; INL, inner nuclear layer; ISH, in situ hybridization; ONL, outer nuclear layer; RPE, retinal pigment epithelium. The paraffin-embedded retina tissue sections were counterstained with Gill hematoxylin to visualize nuclei (blue). Scale bar: 20 µm.

The potential roles of CLRN1 in protein scaffolding and signaling within the retina, as well as its specific molecular impact on Müller glia and photoreceptor neurons are currently unknown. Müller glia are involved in many different aspects of retinal biology, including neuronal development, modulation of neuronal synaptic activity and structural support of photoreceptor cells, and there are species-specific differences between the density and architecture of these cells in the retina.109,110 Previous studies have also suggested a possible function for Müller glia as radial tension sensors, showing that these extraordinary cells are sensitive to mechanically enforced changes in retinal structure.111,112 They have been compared with taut springs, providing mechanical resilience to the retina, and protecting the retina from ripping apart.111 Under normal physiological conditions, the retina is subjected to daily mechanical stress, including during its postnatal expansion. As a tetraspanin, CLRN1 may be involved in maintaining adhesion links between Müller glia and retinal neurons, protecting photoreceptors from mechanical stress, thus enabling their proper function throughout development and with aging.

USH3 patients often display a severe impairment in retinal function at very early stages of the disease, with electroretinogram amplitudes being notably decreased in children as young as 3 to 5 years of age.16 Novel animal models of USH3 that mimic human retinal disease could be used to answer several critical questions with regard to gene therapy approaches. First, is presymptomatic transgene expression of CLRN1 required within photoreceptors, Müller glia, or both cell types to prevent blindness. Second, does ectopic CLRN1 expression in either photoreceptors or Müller glia actually promote degeneration. This could happen if exogenous CLRN1 protein prevents normal USH complexes formation in those cells. Finally, does CLRN1 delivery in adult stages, following symptomatic onset, restore vision loss and slow down the disease progression in USH3 patients. A variety of AAV-based gene therapy vector capsids and promoters targeting either photoreceptors or Müller glia are already available, and more are currently being generated and continuously optimized.23,89,113–116 The development and characterization of large animal models of USH syndrome is urgently needed in the field, as it will contribute greatly to the advancement of our understanding of the pathophysiology of this disease, and critically, provide an opportunity to study the cellular autonomy of retinal degeneration in USH patients.


On the surface, gene replacement appears to be a logical path to treat autosomal recessive retinal pathologies. This has led to the many laboratories and companies moving directly to gene therapeutic strategies to treat certain USH subtypes. The success of this path is yet to be determined. This review has highlighted several hurdles that should be overcome to optimize development of gene therapy towards specific inherited diseases. These fall into 2 general areas, (1) understanding the molecular pathology of the specific disease, and (2) developing gene therapy vectors that safely reach the target cells and express in a manner that effectively corrects biochemical defects without causing collateral damage. In LCA2, the detailed understanding of RPE65 role in vision, the simplicity of the gene structure and availability of both engineered and naturally occurring small (mouse) and large (dog) animal models enabled many researchers to successfully treat these animals, leading to the regulatory approvals of Luxturna. This review and others painfully demonstrate the complexities inherent in understanding the molecular pathophysiology of vision loss secondary to USH disease. Both the lack of solid understanding of the normal function of the associated proteins, and how the omission of these proteins specifically impacts the retina, limit the ability to develop USH therapies. This is true for all forms of USH, but perhaps is most evident for USH3, caused by mutations in an “invisible” CLRN1 protein. A key tool necessary to reveal this information is the generation of suitable animal models displaying a robust visual dysfunction and retinal degeneration phenotype. These models will allow basic scientists to better understand the molecular basis of disease and translational laboratories to develop effective therapies.


The authors thank the Lauhakangas family, whose philanthropic support helped make this research possible. They would also like to acknowledge the support of Usher III Initiative Inc.


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