A protein domain-oriented approach to expand the opportunities of therapeutic exon skipping for USH2A-associated retinitis pigmentosa.

Loss-of-function mutations in USH2A are among the most common causes of syndromic and non-syndromic retinitis pigmentosa (RP). We previously presented skipping of USH2A exon 13 as a promising treatment paradigm for USH2A-associated RP. However, RP-associated mutations are often private, and evenly distributed along the USH2A gene. In order to broaden the group of patients that could benefit from therapeutic exon skipping strategies, we expanded our approach to other USH2A exons in which unique loss-of-function mutations have been reported by implementing a protein domain-oriented dual exon skipping strategy. We first generated zebrafish mutants carrying a genomic deletion of the orthologous exons of the frequently mutated human USH2A exons 30-31 or 39-40 using CRISPR-Cas9. Excision of these in-frame combinations of exons restored usherin expression in the zebrafish retina and rescued the photopigment mislocalization typically observed in ush2a mutants. To translate these findings into a future treatment in humans, we employed in vitro assays to identify and validate antisense oligonucleotides (ASOs) with a high potency for sequence-specific dual exon skipping. Together, the in vitro and in vivo data demonstrate protein domain-oriented ASO-induced dual exon skipping to be a highly promising treatment option for RP caused by mutations in USH2A.

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 .

Zebrafish as a Model to Evaluate a CRISPR/Cas9-Based Exon Excision Approach as a Future Treatment Option for EYS-Associated Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is an inherited retinal disease (IRD) with an overall prevalence of 1 in 4000 individuals. Mutations in EYS (Eyes shut homolog) are among the most frequent causes of non-syndromic autosomal recessively inherited RP and act via a loss-of-function mechanism. In light of the recent successes for other IRDs, we investigated the therapeutic potential of exon skipping for EYS-associated RP. CRISPR/Cas9 was employed to generate zebrafish from which the region encompassing the orthologous exons 37-41 of human EYS (eys exons 40-44) was excised from the genome. The excision of these exons was predicted to maintain the open reading frame and to result in the removal of exactly one Laminin G and two EGF domains. Although the eysΔexon40-44 transcript was found at levels comparable to wild-type eys, and no unwanted off-target modifications were identified within the eys coding sequence after single-molecule sequencing, EysΔexon40-44 protein expression could not be detected. Visual motor response experiments revealed that eysΔexon40-44 larvae were visually impaired and histological analysis revealed a progressive degeneration of the retinal outer nuclear layer in these zebrafish. Altogether, the data obtained in our zebrafish model currently provide no indications for the skipping of EYS exons 37-41 as an effective future treatment strategy for EYS-associated RP.

Opportunities and Pitfalls in Applying Emotion Recognition Software for Persons With a Visual Impairment: Simulated Real Life Conversations.

BACKGROUND: A large part of the communication cues exchanged between persons is nonverbal. Persons with a visual impairment are often unable to perceive these cues, such as gestures or facial expression of emotions. In a previous study, we have determined that visually impaired persons can increase their ability to recognize facial expressions of emotions from validated pictures and videos by using an emotion recognition system that signals vibrotactile cues associated with one of the six basic emotions. OBJECTIVE: The aim of this study was to determine whether the previously tested emotion recognition system worked equally well in realistic situations and under controlled laboratory conditions. METHODS: The emotion recognition system consists of a camera mounted on spectacles, a tablet running facial emotion recognition software, and a waist belt with vibrotactile stimulators to provide haptic feedback representing Ekman's six universal emotions. A total of 8 visually impaired persons (4 females and 4 males; mean age 46.75 years, age range 28-66 years) participated in two training sessions followed by one experimental session. During the experiment, participants engaged in two 15 minute conversations, in one of which they wore the emotion recognition system. To conclude the study, exit interviews were conducted to assess the experiences of the participants. Due to technical issues with the registration of the emotion recognition software, only 6 participants were included in the video analysis. RESULTS: We found that participants were quickly able to learn, distinguish, and remember vibrotactile signals associated with the six emotions. A total of 4 participants felt that they were able to use the vibrotactile signals in the conversation. Moreover, 5 out of the 6 participants had no difficulties in keeping the camera focused on the conversation partner. The emotion recognition was very accurate in detecting happiness but performed unsatisfactorily in recognizing the other five universal emotions. CONCLUSIONS: The system requires some essential improvements in performance and wearability before it is ready to support visually impaired persons in their daily life interactions. Nevertheless, the participants saw potential in the system as an assistive technology, assuming their user requirements can be met.