Generation of Humanized Zebrafish Models for the In Vivo Assessment of Antisense Oligonucleotide-Based Splice Modulation Therapies.

Antisense oligonucleotide (AON)-based splice modulation is the most widely used therapeutic approach to redirect precursor messenger RNA (pre-mRNA) splicing. To study the functional effect of human mutations affecting pre-mRNA splicing for which AON-based splice redirection would be a potential therapeutic option, humanized knock-in animal models are pivotal. A major limitation of using humanized animal models for this purpose is the reported poor recognition of human splice sites by the splicing machineries of other species. To overcome this problem, we provide a detailed guideline for the generation of functional humanized knock-in zebrafish models to assess the effect of mutation-induced aberrant splicing and subsequent AON-based splice modulation therapy .

Antisense oligonucleotide-based treatment of retinitis pigmentosa caused by USH2A exon 13 mutations.

Mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). The two most recurrent mutations in USH2A, c.2299delG and c.2276G > T, both reside in exon 13. Skipping exon 13 from the USH2A transcript presents a potential treatment modality in which the resulting transcript is predicted to encode a slightly shortened usherin protein. Morpholino-induced skipping of ush2a exon 13 in zebrafish ush2armc1 mutants resulted in the production of usherinΔexon 13 protein and a completely restored retinal function. Antisense oligonucleotides were investigated for their potential to selectively induce human USH2A exon 13 skipping. Lead candidate QR-421a induced a concentration-dependent exon 13 skipping in induced pluripotent stem cell (iPSC)-derived photoreceptor precursors from an Usher syndrome patient homozygous for the c.2299delG mutation. Mouse surrogate mQR-421a reached the retinal outer nuclear layer after a single intravitreal injection and induced a detectable level of exon skipping until at least 6 months post-injection. In conclusion, QR-421a-induced exon skipping proves to be a highly promising treatment option for RP caused by mutations in USH2A exon 13.

Poor Splice-Site Recognition in a Humanized Zebrafish Knockin Model for the Recurrent Deep-Intronic c.7595-2144A>G Mutation in USH2A.

The frequent deep-intronic c.7595-2144A>G mutation in intron 40 of USH2A generates a high-quality splice donor site, resulting in the incorporation of a pseudoexon (PE40) into the mature transcript that is predicted to prematurely terminate usherin translation. Aberrant USH2A pre-mRNA splicing could be corrected in patient-derived fibroblasts using antisense oligonucleotides. With the aim to study the effect of the c.7595-2144A>G mutation and USH2A splice redirection on retinal function, a humanized zebrafish knockin model was generated, in which 670 basepairs of ush2a intron 40 were exchanged for 557 basepairs of the corresponding human sequence using an optimized CRISPR/Cas9-based protocol. However, in the retina of adult homozygous humanized zebrafish, only 7.4% ± 3.9% of ush2a transcripts contained the human PE40 sequence and immunohistochemical analyses revealed no differences in the usherin expression and localization between the retina of humanized and wild-type zebrafish larvae. Nevertheless, we were able to partially correct aberrant ush2a splicing using a PE40-targeting antisense morpholino. Our results indicate a clear difference in splice-site recognition by the human and zebrafish splicing machinery. Therefore, we propose a protocol in which the effect of human splice-modulating mutations is studied in a zebrafish-specific cell-based splice assay before the generation of a humanized zebrafish knockin model.

Antisense Oligonucleotide Design and Evaluation of Splice-Modulating Properties Using Cell-Based Assays.

Antisense oligonucleotide (AON)-based splice modulation has been proven to hold great promise as a therapeutic strategy for a number of hereditary conditions. AONs are small modified single-stranded RNA or DNA molecules that are complementary to splice enhancer or silencer target sites. Upon pre-mRNA binding, AONs will prevent or stimulate binding of the spliceosome thereby modulating splicing events. AONs can be designed and applied for different genes and genetic disorders as the specificity depends on their nucleotide sequence. Here we provide a guideline for setting up AON-based splice-modulation experiments by describing a detailed protocol to design and evaluate AONs using a combination of in silico and in vitro analyses.

Eyes shut homolog is important for the maintenance of photoreceptor morphology and visual function in zebrafish.

Mutations in eyes shut homolog (EYS), a gene predominantly expressed in the photoreceptor cells of the retina, are among the most frequent causes of autosomal recessive (ar) retinitis pigmentosa (RP), a progressive retinal disorder. Due to the absence of EYS in several rodent species and its retina-specific expression, still little is known about the exact function of EYS and the pathogenic mechanism underlying EYS-associated RP. We characterized eys in zebrafish, by RT-PCR analysis on zebrafish eye-derived RNA, which led to the identification of a 8,715 nucleotide coding sequence that is divided over 46 exons. The transcript is predicted to encode a 2,905-aa protein that contains 39 EGF-like domains and five laminin A G-like domains, which overall shows 33% identity with human EYS. To study the function of EYS, we generated a stable eysrmc101/rmc101 mutant zebrafish model using CRISPR/Cas9 technology. The introduced lesion is predicted to result in premature termination of protein synthesis and lead to loss of Eys function. Immunohistochemistry on retinal sections revealed that Eys localizes at the region of the connecting cilium and that both rhodopsin and cone transducin are mislocalized in the absence of Eys. Electroretinogram recordings showed diminished b-wave amplitudes in eysrmc101/rmc101 zebrafish (5 dpf) compared to age- and strain-matched wild-type larvae. In addition, decreased locomotor activity in response to light stimuli was observed in eys mutant larvae. Altogether, our study shows that absence of Eys leads to a disorganized retinal architecture and causes visual dysfunction in zebrafish.

Usherin defects lead to early-onset retinal dysfunction in zebrafish.

Mutations in USH2A are the most frequent cause of Usher syndrome and autosomal recessive nonsyndromic retinitis pigmentosa. To unravel the pathogenic mechanisms underlying USH2A-associated retinal degeneration and to evaluate future therapeutic strategies that could potentially halt the progression of this devastating disorder, an animal model is needed. The available Ush2a knock-out mouse model does not mimic the human phenotype, because it presents with only a mild and late-onset retinal degeneration. Using CRISPR/Cas9-technology, we introduced protein-truncating germline lesions into the zebrafish ush2a gene (ush2armc1: c.2337_2342delinsAC; p.Cys780GlnfsTer32 and ush2ab1245: c.15520_15523delinsTG; p.Ala5174fsTer). Homozygous mutants were viable and displayed no obvious morphological or developmental defects. Immunohistochemical analyses with antibodies recognizing the N- or C-terminal region of the ush2a-encoded protein, usherin, demonstrated complete absence of usherin in photoreceptors of ush2armc1, but presence of the ectodomain of usherin at the periciliary membrane of ush2ab1245-derived photoreceptors. Furthermore, defects of usherin led to a reduction in localization of USH2 complex members, whirlin and Adgrv1, at the photoreceptor periciliary membrane of both mutants. Significantly elevated levels of apoptotic photoreceptors could be observed in both mutants when kept under constant bright illumination for three days. Electroretinogram (ERG) recordings revealed a significant and similar decrease in both a- and b-wave amplitudes in ush2armc1 as well as ush2ab1245 larvae as compared to strain- and age-matched wild-type larvae. In conclusion, this study shows that mutant ush2a zebrafish models present with early-onset retinal dysfunction that is exacerbated by light exposure. These models provide a better understanding of the pathophysiology underlying USH2A-associated RP and a unique opportunity to evaluate future therapeutic strategies.

NINL and DZANK1 Co-function in Vesicle Transport and Are Essential for Photoreceptor Development in Zebrafish.

Ciliopathies are Mendelian disorders caused by dysfunction of cilia, ubiquitous organelles involved in fluid propulsion (motile cilia) or signal transduction (primary cilia). Retinal dystrophy is a common phenotypic characteristic of ciliopathies since photoreceptor outer segments are specialized primary cilia. These ciliary structures heavily rely on intracellular minus-end directed transport of cargo, mediated at least in part by the cytoplasmic dynein 1 motor complex, for their formation, maintenance and function. Ninein-like protein (NINL) is known to associate with this motor complex and is an important interaction partner of the ciliopathy-associated proteins lebercilin, USH2A and CC2D2A. Here, we scrutinize the function of NINL with combined proteomic and zebrafish in vivo approaches. We identify Double Zinc Ribbon and Ankyrin Repeat domains 1 (DZANK1) as a novel interaction partner of NINL and show that loss of Ninl, Dzank1 or both synergistically leads to dysmorphic photoreceptor outer segments, accumulation of trans-Golgi-derived vesicles and mislocalization of Rhodopsin and Ush2a in zebrafish. In addition, retrograde melanosome transport is severely impaired in zebrafish lacking Ninl or Dzank1. We further demonstrate that NINL and DZANK1 are essential for intracellular dynein-based transport by associating with complementary subunits of the cytoplasmic dynein 1 motor complex, thus shedding light on the structure and stoichiometry of this important motor complex. Altogether, our results support a model in which the NINL-DZANK1 protein module is involved in the proper assembly and folding of the cytoplasmic dynein 1 motor complex in photoreceptor cells, a process essential for outer segment formation and function.

The pros and cons of vertebrate animal models for functional and therapeutic research on inherited retinal dystrophies.

Over the last decade, huge progress has been made in the understanding of the molecular mechanisms underlying inherited retinal dystrophy (IRD), as well as in the development and implementation of novel therapies, especially in the field of gene therapy. The use of mutant animal models, either naturally occurring or generated by genetic modification, have contributed greatly to our knowledge on IRD. Yet, these mutant animal models do not always mimic the retinal phenotype that is observed in humans with mutations in the orthologous gene, often due to species-specific characteristics of the retina, and/or diverse functions of the gene products in different species. In this manuscript, we compare general and ocular characteristics of a series of widely used vertebrate animal models, i.e. zebrafish, chicken, rodents, cats, dogs, sheep, pigs and monkeys, in terms of genetic architecture and sequence homology, methods to modify genomes, anatomy of the eye, and structural details of the retina. Furthermore, we present an overview of mutant vertebrate animal models that have been used to study or develop treatments for the various genetic subtypes of IRD, and correlate the suitability of these models to the specific characteristics of each animal. Herewith, we provide tools that will help to select the most suitable animal model for specific research questions on IRDs in the future, and thereby assist in an optimal use of animals and resources to further increase our understanding of inherited retinal dystrophies, and develop novel treatments for these disorders.