Models of Retinal Development and Disease

For a model of a human disease to be valid and useful, it is important that key genotypic and phenotypic traits are shared between model system and human. The work in this thesis has been focused on generating new and characterizing spontaneous models of three genetic disorders affecting the retina: retinoblastoma, a childhood cancer with its origin in the fetal retina, Stargardt disease, a juvenile form of macular degeneration, and Bardet-Biedl syndrome, a pleiotropic ciliopathy featuring retinal degeneration. In Paper I, we generated two novel models of MYCN-driven retinoblastoma, in developing chicken retinas and in human retinal organoids, in order to identify its cell of origin and to determine whether MYCN is sufficient in driving tumorigenesis. We found that MYCN was indeed sufficient and that the expression was uniquely tolerated by the cone and horizontal cell progenitor which survived and proliferated anachronistically. The results from our models are in alignment with observations that MYCN-driven retinoblastoma results in a more anaplastic and aggressive form of the cancer and we propose that the fate-restricted progenitor for cones and horizontal cells is its cell of origin. In Paper II, we analyzed the effects of a novel, spontaneous model of ABCA4-mediated Stargardt disease in Labrador retrievers and found that dogs homozygous for the mutation exhibited visual impairment and photoreceptor degeneration. Cone photoreceptors were scarce and the retinal pigment epithelium autofluorescent, indicative of lipofuscin accumulation, a hallmark of Stargardt disease. The phenotype and its etiology in this model are akin to human Stargardt disease. In Paper III, we investigated whether a spontaneous mutation in the TTC8 gene resulted in syndromic Bardet-Biedl syndrome in golden retrievers. We found that, in addition to progressive photoreceptor degeneration and visual impairment, the TTC8 mutation resulted in pleiotropic clinical signs of Bardet-Biedl syndrome with great inter-individual variation, congruent with Bardet-Biedl syndrome in humans. We find that the models described in this thesis are representative of the corresponding genetic disorders in human and that they could be of value for hypothesis generation (in chicken), experimental gene therapy (in dog), and as a human system to test pharmacological interventions (retinal organoids)

Models of Retinal Development and Disease

For a model of a human disease to be valid and useful, it is important that key genotypic and phenotypic traits are shared between model system and human. The work in this thesis has been focused on generating new and characterizing spontaneous models of three genetic disorders affecting the retina: retinoblastoma, a childhood cancer with its origin in the fetal retina, Stargardt disease, a juvenile form of macular degeneration, and Bardet-Biedl syndrome, a pleiotropic ciliopathy featuring retinal degeneration. In Paper I, we generated two novel models of MYCN-driven retinoblastoma, in developing chicken retinas and in human retinal organoids, in order to identify its cell of origin and to determine whether MYCN is sufficient in driving tumorigenesis. We found that MYCN was indeed sufficient and that the expression was uniquely tolerated by the cone and horizontal cell progenitor which survived and proliferated anachronistically. The results from our models are in alignment with observations that MYCN-driven retinoblastoma results in a more anaplastic and aggressive form of the cancer and we propose that the fate-restricted progenitor for cones and horizontal cells is its cell of origin. In Paper II, we analyzed the effects of a novel, spontaneous model of ABCA4-mediated Stargardt disease in Labrador retrievers and found that dogs homozygous for the mutation exhibited visual impairment and photoreceptor degeneration. Cone photoreceptors were scarce and the retinal pigment epithelium autofluorescent, indicative of lipofuscin accumulation, a hallmark of Stargardt disease. The phenotype and its etiology in this model are akin to human Stargardt disease. In Paper III, we investigated whether a spontaneous mutation in the TTC8 gene resulted in syndromic Bardet-Biedl syndrome in golden retrievers. We found that, in addition to progressive photoreceptor degeneration and visual impairment, the TTC8 mutation resulted in pleiotropic clinical signs of Bardet-Biedl syndrome with great inter-individual variation, congruent with Bardet-Biedl syndrome in humans. We find that the models described in this thesis are representative of the corresponding genetic disorders in human and that they could be of value for hypothesis generation (in chicken), experimental gene therapy (in dog), and as a human system to test pharmacological interventions (retinal organoids)

Deletion in the Bardet-Biedl syndrome gene TTC8 results in a syndromic retinal degeneration in dogs

In golden retriever dogs, a 1 bp deletion in the canine TTC8 gene has been shown to cause progressive retinal atrophy (PRA), the canine equivalent of retinitis pigmentosa. In humans, TTC8 is also implicated in Bardet–Biedl syndrome (BBS). To investigate if the affected dogs only exhibit a non-syndromic PRA or develop a syndromic ciliopathy similar to human BBS, we recruited 10 affected dogs to the study. The progression of PRA for two of the dogs was followed for 2 years, and a rigorous clinical characterization allowed a careful comparison with primary and secondary characteristics of human BBS. In addition to PRA, the dogs showed a spectrum of clinical and morphological signs similar to primary and secondary characteristics of human BBS patients, such as obesity, renal anomalies, sperm defects, and anosmia. We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-length TTC8 transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet–Biedl syndrome with heterogeneous clinical signs

MYCN induces cell-specific tumorigenic growth in RB1-proficient human retinal organoid and chicken retina models of retinoblastoma

Retinoblastoma is a rare, intraocular paediatric cancer that originates in the neural retina and is most frequently caused by bi-allelic loss of RB1 gene function. Other oncogenic mutations, such as amplification and increased expression of the MYCN gene, have been found even with proficient RB1 function. In this study, we investigated whether MYCN over-expression can drive carcinogenesis independently of RB1 loss-of-function mutations. The aim was to elucidate the events that result in carcinogenesis and identify the cancer cell-of-origin. We studied the chicken retina, a well-established model for studying retinal neurogenesis, and generated over-expression of MYCN by in ovo electroporation. In parallel, we established an equivalent human stem cell-derived retinal organoid (retinoid) model system. We found that over-expression of MYCN induced tumorigenic growth with high frequency in RB1-proficient chicken retinas and human retinoids. In both systems, the tumorigenic cells expressed markers for undifferentiated cone photoreceptor/horizontal cell progenitors. The over-expression resulted in metastatic retinoblastoma within 7-9 weeks in chicken. MYCN cells could be grown in vitro and, when orthotopically injected, formed tumours that infiltrated the sclera and optic nerve and expressed markers for undifferentiated cones. Investigation of the tumour cell phenotype determined that the potential for neoplastic growth was embryonic stage-dependent and featured a cell-specific resistance to apoptosis in the cone/horizontal cell lineage, but not in ganglion or amacrine cells. We conclude that MYCN over-expression is sufficient to drive tumorigenesis and that a cell-specific resistance to apoptosis in the cone/horizontal cell lineage mediates the cancer phenotype

Deletion in the Bardet-Biedl Syndrome Gene TTC8 Results in a Syndromic Retinal Degeneration in Dogs

In golden retriever dogs, a 1 bp deletion in the canineTTC8gene has been shown to cause progressive retinal atrophy (PRA), the canine equivalent of retinitis pigmentosa. In humans,TTC8is also implicated in Bardet-Biedl syndrome (BBS). To investigate if the affected dogs only exhibit a non-syndromic PRA or develop a syndromic ciliopathy similar to human BBS, we recruited 10 affected dogs to the study. The progression of PRA for two of the dogs was followed for 2 years, and a rigorous clinical characterization allowed a careful comparison with primary and secondary characteristics of human BBS. In addition to PRA, the dogs showed a spectrum of clinical and morphological signs similar to primary and secondary characteristics of human BBS patients, such as obesity, renal anomalies, sperm defects, and anosmia. We used Oxford Nanopore long-read cDNA sequencing to characterize retinal full-lengthTTC8transcripts in affected and non-affected dogs, the results of which suggest that three isoforms are transcribed in the retina, and the 1 bp deletion is a loss-of-function mutation, resulting in a canine form of Bardet-Biedl syndrome with heterogeneous clinical signs

An ABCA4 loss-of-function mutation causes a canine form of Stargardt disease

Autosomal recessive retinal degenerative diseases cause visual impairment and blindness in both humans and dogs. Currently, no standard treatment is available, but pioneering gene therapy-based canine models have been instrumental for clinical trials in humans. To study a novel form of retinal degeneration in Labrador retriever dogs with clinical signs indicating cone and rod degeneration, we used whole-genome sequencing of an affected sib-pair and their unaffected parents. A frameshift insertion in the ATP binding cassette subfamily A member 4 (ABCA4) gene (c.4176insC), leading to a premature stop codon in exon 28 (p.F1393Lfs*1395), was identified. In contrast to unaffected dogs, no full-length ABCA4 protein was detected in the retina of an affected dog. The ABCA4 gene encodes a membrane transporter protein localized in the outer segments of rod and cone photoreceptors. In humans, the ABCA4 gene is associated with Stargardt disease (STGD), an autosomal recessive retinal degeneration leading to central visual impairment. A hallmark of STGD is the accumulation of lipofuscin deposits in the retinal pigment epithelium (RPE). The discovery of a canine homozygous ABCA4 loss-of-function mutation may advance the development of dog as a large animal model for human STGD. Author summary Stargardt disease (STGD) is the most common inherited retinal disease causing visual impairment and blindness in children and young adults, affecting 1 in 8-10 thousand people. For other inherited retinal diseases, the dog has become an established comparative animal model, both for identifying the underlying genetic causes and for developing new treatment methods. To date, there is no standard treatment for STGD and the only available animal model to study the disease is the mouse. As a nocturnal animal, the morphology of the mouse eye differs from humans and therefore the mouse model is not ideal for developing methods for treatment. We have studied a novel form of retinal degeneration in Labrador retriever dogs showing clinical signs similar to human STGD. To investigate the genetic cause of the disease, we used whole-genome sequencing of a family quartet including two affected offspring and their unaffected parents. This led to the identification of a loss-of-function mutation in the ABCA4 gene. The findings of this study may enable the development of a canine model for human STGD