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

Introduction Usher syndrome (USH) represents a group of genetically heterogenous autosomal recessive disorders, characterized by combined vision and sensorineural hearing loss, in some cases vestibular dysfunction.1–3 It primarily affects the light-sensitive photoreceptor cells retina auditory hair cochlea. Vision loss all is progressive, manifests as retinitis pigmentosa (RP), gradual degeneration cells.4 Peripheral rod function lost first, leading to night blindness constricted visual fields, followed death cones severe impairment. There are currently no drugs or biological therapies proven be effective treating USH syndrome. Cochlear implantation, which bypasses damaged stimulates primary neurons directly, an approach alleviate impairment patients. However, there remedy for progressive retina, thus critical unmet need exists develop therapeutic strategies prevent The generation animal models that faithfully mimic human disorder, timing gene therapy interventions, identification correct target patient selection key factors successfully reaching this goal. was named after Dr Charles Howard Usher, Scottish ophthalmologist who described 69 patients with disease 1914.5 As commonly case medicine, first much earlier (1858) brothers suffering from deafness von Gräfe6 further his student, Liebreich,7 3 years later larger population From its discovery until recently, subgroups were delineated clinical characteristics.8 Now recognized symptomology classified genotyping. To date, at least 10 causative genes associated syndrome, summarized previous reviews.1,2,9 In general, based on diverse symptoms, particularly onset severity presence dysfunction, grouped into subtypes. Type I severe-to-profound prelingual abnormalities prepubescent RP symptoms.10,11 USH1 results mutations MYO7A (USH1B), HARMONIN (USH1C), CDH23 (USH1D), PCDH15 (USH1F), SANS (USH1G), CIB2 (USH1J). Patients type II display moderate-to-severe congenital without account >50% cases. Their symptoms generally begin second decade.12 USH2 caused USH2A (USH2A), ADGRV1 (USH2C), WHRN (USH2D). III CLRN1 (USH3A) have postlingual variable rapidly progresses legal fourth decade life.13–17 Mutations HARS (Histidyl-tRNA synthetase) ultra-rare form USH3B.18,19 Other been atypical USH, including: CEP250 CEP78, encoding members CEP family centrosome-associated proteins; ESPN, F-actin cross-linker espin; ARSG, arylsulfatase G enzyme.20 Gene Therapy Syndrome: Current Challenges success Leber Congenital Amaurosis (LCA2) due defects RPE65 subsequent FDA EMA approval Luxturna (voretigene neparvovec) sparked hope many other retinal (and neurological) diseases may also treated using vector-based delivery approach.21,22 exception several laboratories embarked transfer studies models. Conventional recombinant adeno-associated viral vectors (AAVs) contain single-stranded DNA expression cassette, includes cell-specific ubiquitous promoter, transgene polyadenylation signal, flanked palindromic inverted terminal repeats both ends. contrast systems, AAV low immunogenicity (especially when given subretinally), long-lasting postmitotic their ability persist episomes, efficiently transduce photoreceptors pigmented epithelium (RPE), where most RP-associated expressed.23 Overall, hold tremendous potential numerous challenges field, become apparent acknowledging disease-specific parameters AAV-based augmentation LCA. These include availability small large RPE65-deficiency displaying robust phenotype, slow degenerative component accompanying marked window treatment, lack detrimental effects following long-term AAV-mediated overexpression RPE.24 ocular molecular basis dysfunction LCA2 well understood. Moreover, causes substantial murine canine models, demonstrated significantly reduced nonrecordable electroretinograms.25–27 contrasts sharply remarkable preservation architecture, has allowed testing effects.28–30 protein 65 kDa retinoid isomerohydrolase, plays essential role cycle converting all-trans-retinyl ester 11-cis-retinol RPE cells.31–33 11-cis chromophore deficiency outer segments can corrected restoring enzyme RPE, either promoter modified version chicken-beta actin promoter. Furthermore, one interdigitates 20 40 apical interface, amplifying while minimizing downside nonuniform neighboring cells. Subretinal injection AAV2 vector delivering wild-type cDNA dog produced beneficial protection especially treatment initiated early.29,30,34,35 Ultimately, successful restoration model paved way towards patients.36–40 Unlike RPE65-associated disease, majority engineered mouse do not exhibit although they loss.41,42 Some these reported only aging.42 suggested phenotype could result distinct species-specific cell pattern morphologic differences between photoreceptors, example, mice well-developed, actin-filled calyceal processes, subcellular structures extending inner frogs, pigs, zebrafish, primates.43,44 absence severely limited perform therapeutic, preclinical proof-of-concept replacement, editing, molecule used preserve positive results.9,45–53 recent, in-depth reviews therapies.9,54,55 data, nature motivated few companies empirically study treatments subjects. One example lentiviral-mediated USH1B.56,57 This shown melanosome mislocalization opsin accumulation connecting cilium Myo7a-deficient shaker1 USH1B.57 packaging capacity compared lentiviral (4.7 vs. 8.5 kb). cDNA, expressed cells, too (~6.7 kb) packaged vectors.58 Therefore, equine infectious anemia virus-based (equine virus-CMV-MYO7A, UshStat) subretinally delivered USH1B goal down progression (ClinicalTrials.gov identifier: NCT01505062). Long-term safety evaluation received UshStat being performed trial (NCT02065011, pending). Another (UshTher) underway, uses dual express subretinal (https://cordis.europa.eu/project/id/754848/it). Dual take advantage 2 genomes containing 5? 3? half undergo intermolecular concatamerization, homologous recombination reconstitute whole cassette.59,60 clinically data trials. genes, (cDNA~15.6 kb), benefit genome editing approaches, such CRISPR-Cas9-sgRNAs, those promote exon skipping restore functional restoration.61–66 A splicing-modulating antisense oligonucleotide designed cause in-frame mutated 13 disease-associated transcripts shortened but usherin protein, localizes correctly cells.66–68 Interestingly, recent zebrafish early-onset indicating represent useful tools investigate pathophysiology underlying test strategies.69,70 Morpholino oligonucleotides mediate ush2a rescue model.66 mutation-specific strategy evaluated (QR-421a) administered through intravitreal injections, early evidence efficacy 3-month interim analysis (phase 1/2 Stellar QR-421a, ProQR Therapeutics, Clinicaltrials.gov ID: NCT03780257). Critical future approaches characterization novel disease. will serve platforms therapies, including viral-based strategies, gain insight mechanisms pig USH1C lacking harmonin, (R31X), recently deafness, changes function.71 estimated prevalence ranging 4 17 per 100,000 individuals worldwide.72,73 Approximately 98% belong 1 contrast, USH3 relatively rare, accounting ~2% cases.74 it prevalent Finnish population, 40% cases, segregates high degree Ashkenazi-Jewish descent.16,17,74 Through funding initiative, team researchers brought together 2007 uncover roles sensory organs pathology ensues defective. Several developed sustained effort, ultimate USH3A An offshoot effort resulted NIH-funded grants specifically directed understanding complex rest review highlight important findings research, discuss developing potentially Protein Network considerable amount research Fluorescence colocalization tissue sections different species reciprocal pull-down assays suggest proteins encoded interact each organize complexes within specific regions cells.75 They highly dynamic networks, consist classes transport, adhesion, signaling processes types. actin-based motor myosin VIIa (MYO7A, USH1B); cell-adhesion transmembrane cadherin 23 (CDH23, USH1D), protocadherin 15 (PCDH15, USH1F), (USH2A); very-large protein-coupled receptor-1 VLGR1 (ADGRV1, GPR98, USH2C); calcium integrin binding (CIB2, USH1J); PDZ-domain scaffolding harmonin (USH1C) whirlin (WHRN, USH2D); ankyrin SAM-domain (USH1G); tetraspanin-like clarin-1 (CLRN1, USH3A). found concentrate specialized cilium, periciliary membrane.2 detected ribbon synapses enable rapid release neurotransmitters necessary frequency transmission signals cells.76 system often served mirror functions retina. cochlea, bundle, mechanosensory antenna rich stereocilia projecting surface elegant transient permanent extracellular filamentous links stereocilia, Sans, anchor intracellular core.77–80 adult stages, tip links, act similar gating springs open mechanoelectrical transduction ion channels response mechanical stimuli provided sound waves.81,82 fragile actin-rich macaque, humans, play development maintenance.43,83 Similar interstereociliary stabilize membrane-membrane connection sites linking segments, allowing them withstand daily stress.43 Consistent possibility, interactions microvilli absorptive intestinal cells.84,85 USH1, usherin, VLGR1, mainly located ridge membrane region surrounding provide structural support participate vesicle docking plasma during translocation segment.63,86 Pathogenic particular alter localization network, property exploited evaluate experiments models.87 Considering transport maintenance, careful dosing choice regulatable promoters needed ensure vector-mediated mimics endogenous levels does lead structure function.88 use penetrating capsids higher efficiency targeting surgically safer approach, reducing concentration avoiding effects.89,90 CLRN1: “Invisible” Tetraspanin Unknown Biological Function major challenge field lies difficulty reliably detecting epitope availability, consequence expression. aspect, thoroughly validated antibody reagents proper negative controls certain generated conflicting information regarding cellular true CLRN1, glycoprotein whose unknown.91 main isoform encodes 232 amino acid contains single glycosylation site asparagine 48 (N48) loop PDZ motif C-terminal end.91,92 vitro hemagglutinin (HA)-epitope tagged HEK concentrates forms CLRN1-enriched microdomains, recruiting involved cell-adhesion, focal tight junctions, organizing cytoskeleton.93 PCDH15.47,94Clrn1 knockout N48K knock-in disorganized F-actin-rich cochlea maintain integrity bundle.95–97 established bundle protein.98 synaptic cells.47,94 Three postnatal Clrn1 cochlear delivery, preserved.47–49 proof principle raise successful, providing before significant damage occurs. Studies aimed define eye both, depending method (in situ hybridization mRNA immunostaining protein).97,99–101 Localization confounded RNA probes antibodies used, variability sample processing conditions, analyzed. study, transiently (ISH), consistent progenitor Müller glia expression.97 By rodless aged rd1 laser capture microdissection, showed retinas confined nuclear layer, detectable RT-PCR. same year, another custom made anti-CLRN1 antibody.101 retina.99 concluded remains below detection immunostaining.47,49,97,100 Recently, we N-terminus epitope-tagged facilitate affinity anti-HA antibody.100 We noted background labeling HA-tagged C57BL/6J controls. Importantly, western blot homogenates, continuously adulthood.100 viral-mediated approach.100,102 single-cell sequencing (scRNAseq) technology unprecedented opportunity firmly map multiple species, complementary tool immunodetection.103,104 our published scRNAseq sets, bypassed experiments, convincingly present human, non-human primates.100 enrichment corroborated samples studies.105–108 sets (USH1G) glia, supporting consider involvement types disease.108 sensitive RNAscope ISH assay tissue, data.100 No signal sections, confirming specificity assay.100 examined chromogenic assay. determined abundantly development, fully developed, concentrating middle part layer piglet (Fig. 1). Taken together, solid complexities inherent potentially, biology dysfunction. completely rule out possibility might very levels, undetectable current technologies.Figure 1: Detection day P2 (red punctate staining). GCL indicates ganglion layer; INL, ISH, hybridization; ONL, pigment epithelium. paraffin-embedded counterstained Gill hematoxylin visualize nuclei (blue). Scale bar: µm.The impact unknown. aspects biology, neuronal modulation activity density architecture retina.109,110 Previous possible radial tension sensors, showing extraordinary mechanically enforced structure.111,112 taut springs, resilience protecting ripping apart.111 Under normal physiological subjected stress, expansion. tetraspanin, maintaining adhesion neurons, enabling throughout aging. stages electroretinogram amplitudes notably decreased children young 5 age.16 Novel answer questions regard approaches. First, presymptomatic required blindness. Second, ectopic actually degeneration. happen if exogenous prevents formation Finally, symptomatic onset, variety already available, more optimized.23,89,113–116 urgently contribute greatly advancement critically, autonomy Conclusions On surface, replacement appears logical path treat pathologies. led moving directly yet determined. highlighted hurdles should overcome optimize inherited diseases. fall general areas, (1) (2) safely reach manner effectively corrects biochemical causing collateral damage. LCA2, detailed vision, simplicity naturally occurring (mouse) (dog) enabled animals, regulatory approvals Luxturna. others painfully demonstrate secondary Both proteins, how omission impacts limit therapies. perhaps evident USH3, “invisible” protein. reveal suitable phenotype. allow basic scientists better understand translational