Gene augmentation therapy attenuates retinal degeneration in a knockout mouse model of Fam161a retinitis pigmentosa.

Photoreceptor cell degeneration and death is the major hallmark of a wide group of human blinding diseases including age-related macular degeneration and inherited retinal diseases such as retinitis pigmentosa. In recent years, inherited retinal diseases have become the "testing ground" for novel therapeutic modalities, including gene and cell-based therapies. Currently there is no available treatment for retinitis pigmentosa caused by FAM161A biallelic pathogenic variants. In this study, we injected an adeno-associated virus encoding for the longer transcript of mFam161a into the subretinal space of P24-P29 Fam161a knockout mice to characterize the safety and efficacy of gene augmentation therapy. Serial in vivo assessment of retinal function and structure at 3, 6, and 8 months of age using the optomotor response test, full-field electroretinography, fundus autofluorescence, and optical coherence tomography imaging as well as ex vivo quantitative histology and immunohistochemical studies revealed a significant structural and functional rescue effect in treated eyes accompanied by expression of the FAM161A protein in photoreceptors. The results of this study may serve as an important step toward future application of gene augmentation therapy in FAM161A-deficient patients by identifying a promising isoform to rescue photoreceptors and their function.

Morphological and Functional Comparison of Mice Models for Retinitis Pigmentosa.

Retinitis pigmentosa (RP) is the predominant form of inherited retinal degenerations (IRDs) caused by abnormalities and loss of photoreceptor cells ensuing diminishment of vision. RP is a heterogenous genetic disorder associated with mutations in over 80 genes, showing various inheritance patterns. Laboratory mouse models are important for our understanding of disease mechanisms, modifier effects, and development of therapeutic modalities. In this review, we have summarized a comprehensive comparison of our previously reported Fam161a knockout (KO) mouse model with other well-studied RP mouse models, Fam161aGT/GT, Pde6brd1, Nr2e3rd7, Rpgrrd9, and Pde6brd10 using structural and functional analysis of the retina. Fam161atm1b/tm1b mouse models are important for developing novel therapies and mainly AAV-based gene therapy and translational read-through-inducing drugs.

Translational Read-Through Drugs (TRIDs) Are Able to Restore Protein Expression and Ciliogenesis in Fibroblasts of Patients with Retinitis Pigmentosa Caused by a Premature Termination Codon in FAM161A.

Ataluren and Gentamicin are translational readthrough drugs (TRIDs) that induce premature termination codon (PTC) readthrough, resulting in the production of full-length proteins that usually harbor a single missense substitution. FAM161A is a ciliary protein which is expressed in photoreceptors, and pathogenic variants in this gene cause retinitis pigmentosa (RP). Applying TRIDs on fibroblasts from RP patients due to PTC in the FAM161A (p.Arg523*) gene may uncover whether TRIDs can restore expression, localization and function of this protein. Fibroblasts from six patients and five age-matched controls were starved prior to treatment with ataluren or gentamicin, and later FAM161A expression, ciliogenesis and cilia length were analyzed. In contrast to control cells, fibroblasts of patients did not express the FAM161A protein, showed a lower percentage of ciliated cells and grew shorter cilia after starvation. Ataluren and Gentamicin treatment were able to restore FAM161A expression, localization and co-localization with α-tubulin. Ciliogenesis and cilia length were restored following Ataluren treatment almost up to a level which was observed in control cells. Gentamicin was less efficient in ciliogenesis compared to Ataluren. Our results provide a proof-of-concept that PTCs in FAM161A can be effectively suppressed by Ataluren or Gentamicin, resulting in a full-length functional protein.

Interactions between C8orf37 and FAM161A, Two Ciliary Proteins Essential for Photoreceptor Survival.

Mutations in C8orf37 cause Bardet-Biedl syndrome (BBS), retinitis pigmentosa (RP), and cone-rod dystrophy (CRD), all manifest in photoreceptor degeneration. Little is known about which proteins C8orf37 interacts with to contribute to photoreceptor survival. To determine the proteins that potentially interact with C8orf37, we carried out a yeast two-hybrid (Y2H) screen using C8orf37 as a bait. FAM161A, a microtubule-binding protein localized at the photoreceptor cilium required for photoreceptor survival, was identified as one of the preys. Double immunofluorescence staining and proximity ligation assay (PLA) of marmoset retinal sections showed that C8orf37 was enriched and was co-localized with FAM161A at the ciliary base of photoreceptors. Epitope-tagged C8orf37 and FAM161A, expressed in HEK293 cells, were also found to be co-localized by double immunofluorescence staining and PLA. Furthermore, interaction domain mapping assays identified that the N-terminal region of C8orf37 and amino acid residues 341-517 within the PFAM UPF0564 domain of FAM161A were critical for C8orf37-FAM161A interaction. These data suggest that the two photoreceptor survival proteins, C8orf37 and FAM161A, interact with each other which may contribute to photoreceptor health.

FAM161A and TTC8 are Differentially Expressed in Non-Allelelic Early Onset Retinal Degeneration.

Ciliary genes FAM161A and TTC8 have been implicated in retinal degeneration (RD) in humans and in dogs. The identification of FAM161A and TTC8 mutations in canine RD is exciting as there is the potential to develop novel large animal models for RD. However, the disease phenotypes in the dog and the roles of abnormal genes in disease pathology have yet to be fully characterized. The present study evaluated the expression patterns of FAM161A and TTC8 during normal retinal development in dogs, and in three non-allelic, early onset canine RD models at critical time points of the disease: RCD1, XLPRA2 and ERD. Both genes were differentially expressed in RCD1 and ERD, but not in XLPRA2. These results add evidence to the hypothesis that (a) mutations in many retinal genes have a cascade effect on the expression of multiple, possibly unrelated genes and (b) a large number and wide range of genes probably contribute to RD in general.

Fly Fam161 is an essential centriole and cilium transition zone protein with unique and diverse cell type-specific localizations.

Family with sequence similarity 161 (Fam161) is an ancient family of microtubule-binding proteins located at the centriole and cilium transition zone (TZ) lumen that exhibit rapid evolution in mice. However, their adaptive role is unclear. Here, we used flies to gain insight into their cell type-specific adaptations. Fam161 is the sole orthologue of FAM161A and FAM161B found in flies. Mutating Fam161 results in reduced male reproduction and abnormal geotaxis behaviour. Fam161 localizes to sensory neuron centrioles and their specialized TZ (the connecting cilium) in a cell type-specific manner, sometimes labelling only the centrioles, sometimes labelling the centrioles and cilium TZ and sometimes labelling the TZ with varying lengths that are longer than other TZ proteins, defining a new ciliary compartment, the extra distal TZ. These findings suggest that Fam161 is an essential centriole and TZ protein with a unique cell type-specific localization in fruit flies that can produce cell type-specific adaptations.

Exonic Short Interspersed Nuclear Element Insertion in FAM161A Is Associated with Autosomal Recessive Progressive Retinal Atrophy in the English Shepherd.

Progressive retinal atrophies (PRAs) are a genetically heterogeneous group of inherited eye diseases that affect over 100 breeds of dog. The initial clinical sign is visual impairment in scotopic conditions, as a consequence of rod photoreceptor cell degeneration. Photopic vision degeneration then follows, due to progression of the disease to the cone photoreceptors, and ultimately results in complete blindness. Two full-sibling English Shepherds were diagnosed with PRA at approximately 5 years old and tested clear of all published PRA genetic variants. This study sought to identify the novel PRA-associated variant segregating in the breed. We utilised a combined approach of whole genome sequencing of the probands and homozygosity mapping of four cases and 22 controls and identified a short interspersed nuclear element within an alternatively spliced exon in FAM161A. The XP_005626197.1 c.17929_ins210 variant was homozygous in six PRA cases and heterozygous or absent in control dogs, consistent with a recessive mode of inheritance. The insertion is predicted to extend exon 4 by 39 aberrant amino acids followed by an early termination stop codon. PRA is intractable to treatment, so the development of a genetic screening test, based on the associated variant, is significant, because it provides dog breeders/owners with a means of reducing the frequency of the disease variant within this breed as well as minimising the risk of breeding puppies that will develop this blinding disease.

Retinal Structure and Function in a Knock-in Mouse Model for the FAM161A-p.Arg523∗ Human Nonsense Pathogenic Variant.

Purpose: Pathogenic variants in FAM161A are the most common cause of retinitis pigmentosa in Israel. Two founder pathogenic variants explain the vast majority of cases of Jewish origin, 1 being a nonsense variant (p.Arg523∗). The aim of this study was to generate a knock-in (KI) mouse model harboring the corresponding p.Arg512∗ pathogenic variant and characterize the course of retinal disease. Design: Experimental study of a mouse animal model. Subjects/Participants/Controls: A total of 106 Fam161a knock-in mice and 29 wild-type mice with C57BL/6J background particiapted in this study. Methods: Homozygous Fam161a p.Arg512∗ KI mice were generated by Cyagen Biosciences. Visual acuity (VA) was evaluated using optomotor tracking response and retinal function was assessed by electroretinography (ERG). Retinal structure was examined in vivo using OCT and fundus autofluorescence imaging. Retinal morphometry was evaluated by histologic and immunohistochemical (IHC) analyses. Main Outcome Measures: Visual and retinal function assessments, clinical imaging examinations, quantitative histology, and IHC studies of KI as compared with wild-type (WT) mice retinas. Results: The KI model was generated by replacing 3 bp, resulting in p.Arg512∗. Homozygous KI mice that had progressive loss of VA and ERG responses until the age of 18 months, with no detectable response at 21 months. OCT showed complete loss of the outer nuclear layer at 21 months. Fundus autofluorescence imaging revealed progressive narrowing of blood vessels and formation of patchy hyper-autofluorescent and hypo-autofluorescent spots. Histologic analysis showed progressive loss of photoreceptor nuclei. Immunohistochemistry staining showed Fam161a expression mainly in photoreceptors cilia and the outer plexiform layer (OPL) in WT mice retinas, whereas faint expression was evident mainly in the cilia and OPL of KI mice. Conclusions: The Fam161a - p.Arg512∗ KI mouse model is characterized by widespread retinal degeneration with relatively slow progression. Surprisingly, disease onset is delayed and progression is slower compared with the previously reported knock-out model. The common human null mutation in the KI mouse model is potentially amenable for correction by translational read-through-inducing drugs and by gene augmentation therapy and RNA editing, and can serve to test these treatments as a first step toward possible application in patients. Financial Disclosures: The author(s) have no proprietary or commercial interest in any materials discussed in this article.

Structural bioinformatics predicts that the Retinitis Pigmentosa-28 protein of unknown function FAM161A is a homologue of the microtubule nucleation factor Tpx2.

Background: FAM161A is a microtubule-associated protein conserved widely across eukaryotes, which is mutated in the inherited blinding disease Retinitis Pigmentosa-28. FAM161A is also a centrosomal protein, being a core component of a complex that forms an internal skeleton of centrioles. Despite these observations about the importance of FAM161A, current techniques used to examine its sequence reveal no homologies to other proteins. Methods: Sequence profiles derived from multiple sequence alignments of FAM161A homologues were constructed by PSI-BLAST and HHblits, and then used by the profile-profile search tool HHsearch, implemented online as HHpred, to identify homologues. These in turn were used to create profiles for reverse searches and pair-wise searches. Multiple sequence alignments were also used to identify amino acid usage in functional elements. Results: FAM161A has a single homologue: the targeting protein for Xenopus kinesin-like protein-2 (Tpx2), which is a strong hit across more than 200 residues. Tpx2 is also a microtubule-associated protein, and it has been shown previously by a cryo-EM molecular structure to nucleate microtubules through two small elements: an extended loop and a short helix. The homology between FAM161A and Tpx2 includes these elements, as FAM161A has three copies of the loop, and one helix that has many, but not all, properties of the one in Tpx2. Conclusions: FAM161A and -its homologues are predicted to be a previously unknown variant of Tpx2, and hence bind microtubules in the same way. This prediction allows precise, testable molecular models to be made of FAM161A-microtubule complexes.

Microglia Analysis in Retinal Degeneration Mouse Models.

Microgliosis is a hallmark of degenerative processes in the retina. Reactive microglia migrate to the photoreceptor layer and the subretinal space during outer retinal degeneration. This process creates a toxic milieu where reactive microglia and dying photoreceptors recruit additional reactive phagocytes. This results in the release of a multitude of proinflammatory factors which accelerate photoreceptor demise. In this chapter, we outline in detail how to monitor microgliosis in the Fam161a-deficient mouse model of Retinitis Pigmentosa by performing immunohistochemical stainings of retinal cryosections and flat mounts using the marker Iba1. This protocol will serve as a guideline in evaluating microglia reactivity and localization in various mouse models of retinal degeneration.

Ocular Phenotype of a Family with FAM161A-associated Retinal Degeneration.

BACKGROUND: Characterization of retinal degeneration (RD) using high-resolution retinal imaging and exome sequencing may identify phenotypic features that correspond with specific genetic defects. MATERIALS AND METHODS: Six members from a non-consanguineous Indian family (three affected siblings, their asymptomatic parents and an asymptomatic child) were characterized clinically, using visual acuity, perimetry, full-field electroretinography (ERG), optical coherence tomography and cone structure as outcome measures. Cone photoreceptors were imaged in the proband using adaptive optics scanning laser ophthalmoscopy. The exome was captured using Nimblegen SeqCap EZ V3.0 probes and sequenced using lllumina HiSeq. Reads were mapped to reference hg19. Confirmation of variants and segregation analysis was performed using dideoxy sequencing. RESULTS: Analysis of exome variants using exomeSuite identified five homozygous variants in four genes known to be associated with RD. Further analysis revealed a homozygous nonsense mutation, c.1105 C > T, p.Arg335Ter, in the FAM161A gene segregating with RD. Three additional variants were found to occur at high frequency. Affected members showed a range of disease severity beginning at different ages, but all developed severe visual field and outer retinal loss. CONCLUSIONS: Exome analysis revealed a nonsense homozygous mutation in FAM161A segregating with RD with severe vision loss and a range of disease onset and progression. Loss of outer retinal structures demonstrated with high-resolution retinal imaging suggests FAM161A is important for normal photoreceptor structure and survival. Exome sequencing may identify causative genetic variants in autosomal recessive RD families when other genetic test strategies fail to identify a mutation.