Hemant Khanna

Ciliopathies are a class of inherited severe human disorders that occur due to defective formation or function of cilia. The RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein1-like) gene encodes for a ciliary protein involved in regulating cilia formation and function. Mutations in RPGRIP1L cause ciliopathies associated with severe embryonic defects, such as Meckel-Gruber Syndrome (MKS). Here we report RPGRIP1L mutation analysis in a family diagnosed with MKS. The clinical manifestations of the fetus included thoraco-lumbar open neural tube defect with associated Chiari type II malformation and hydrocephalus, bilateral club feet, and single right kidney/ureter. Analysis of the parental DNA samples revealed that the father carried a previously reported mutation R1236C/+ whereas the mother had a novel splice site mutation IVS6+1 G > A/+ in RPGRIP1L. The splice site mutation resulted in the exclusion of in-frame exon 6 of RPGRIP1L (RPGRIP1L-∆Ex6) but expressed a stable protein in fibroblasts derived from the parents’ skin biopsies. The GFP-RPGRIP1L-∆Ex6 mutant protein exhibited relatively reduced ciliary localization in transiently-transfected cultured RPE-1 cells. Taken together, this study identifies a novel RPGRIP1L variant RPGRIP1L-∆Ex6, which in combination with RPGRIP1L-R1236C is associated with MKS. We also suggest that the deletion of exon 6 of RPGRIP1L leads to reduced ciliary localization of RPGRIP1L, indicating a plausible mechanism of associated disease.

The eye is at the forefront of developing therapies for genetic diseases. With the FDA approval of the first gene-therapy drug for a form of congenital blindness, numerous studies have been initiated to develop gene therapies for other forms of eye diseases. These examinations have revealed new information about the benefits as well as restrictions to using drug-delivery routes to the different parts of the eye. In this article, we will discuss a brief history of gene therapy and its importance to the eye and ocular delivery landscape that is currently being investigated, and provide insights into their advantages and disadvantages. Efficient delivery routes and vehicle are crucial for an effective, safe, and longer-lasting therapy.

Mutations in retinitis pigmentosa GTPase regulator (RPGR) cause severe retinal ciliopathy, X-linked retinitis pigmentosa. Although two major alternatively spliced isoforms, RPGR^ex1-19 and RPGR^ORF15, are expressed, the relative importance of these isoforms in disease pathogenesis is unclear. Here, we analyzed fibroblast samples from eight patients and found that all of them form longer cilia than normal controls, albeit to different degrees. Although all mutant RPGR^ORF15 messenger RNAs (mRNAs) are unstable, their steady-state levels were similar or higher than those in the control cells, suggesting there may be increased transcription. Three of the fibroblasts that had higher levels of mutant RPGR^ORF15 mRNA also exhibited significantly higher levels of RPGR^ex1-19 mRNA. Four samples with unaltered RPGR^ex1-19 levels carried mutations in RPGR^ORF15 that resulted in this isoform being relatively less stable. Thus, in all cases, the RPGR^ex1-19/RPGR^ORF15 isoform ratio was increased, and this was highly correlative to the cilia extension defect. Moreover, overexpression of RPGR^ex1-19 (mimicking the increase in RPGR^ex1-19 to RPGR^ORF15 isoform ratio) or RPGR^ORF15 (mimicking reduction of the ratio) resulted in significantly longer or shorter cilia, respectively. Notably, the cilia length defect appears to be attributable to both the loss of the wild-type RPGR^ORF15 protein and to the higher levels of the RPGR^ex1-19 isoform, indicating that the observed defect is due to the altered isoform ratios. These results suggest that maintaining the optimal RPGR^ex1-9 to RPGR^ORF15 ratio is critical for cilia growth and that designing strategies that focus on the best ways to restore the RPGR^ex1-19/RPGR^ORF15 ratio may lead to better therapeutic outcomes.

Fibrocellular membrane or epiretinal membrane (ERM) forms on the surface of the inner limiting membrane (ILM) in the inner retina and alters the structure and function of the retina. ERM formation is frequently observed in ocular inflammatory conditions, such as proliferative diabetic retinopathy (PDR) and retinal detachment (RD). Although peeling of the ERM is used as a surgical intervention, it can inadvertently distort the retina. Our goal is to design alternative strategies to tackle ERMs. As a first step, we sought to determine the composition of the ERMs by identifying the constituent cell-types and gene expression signature in patient samples. Using ultrastructural microscopy and immunofluorescence analyses, we found activated microglia, astrocytes, and Müller glia in the ERMs from PDR and RD patients. Moreover, oxidative stress and inflammation associated gene expression was significantly higher in the RD and PDR membranes as compared to the macular hole samples, which are not associated with inflammation. We specifically detected differential expression of hypoxia inducible factor 1-α (HIF1-α), proinflammatory cytokines, and Notch, Wnt, and ERK signaling pathway-associated genes in the RD and PDR samples. Taken together, our results provide new information to potentially develop methods to tackle ERM formation.

X-linked retinitis pigmentosa 2 (XLRP2) patients and Rp2^null mice exhibit severe cone photoreceptor degeneration. However, due to the paucity of cones in mammalian model systems, it is not clear how cones respond to the loss of RP2. Here we have used the Nrl^-/- mice, which develop a rodless and short wavelength (S) opsin-containing cone-only retina, to generate Rp2^null::Nrl^-/- double knock out (Rp2-DKO) mice. We found that the ciliary axoneme and the outer segments (OSs) of the cones were significantly longer with disorganized membrane infoldings as compared to the Nrl^-/- mice. Additionally, we found misregulation in the expression of the genes related to ophthalmic disease, cell trafficking, and stress-response in the Rp2-DKO mice prior to the onset of cone degeneration. Surprisingly, the loss of RP2 did not affect progressive photoreceptor dysfunction of the Nrl^-/- mice and the trafficking of S opsin. Our data suggest that RP2 is a negative regulator of cone OS length but does not affect S-opsin trafficking and S-cone function. Our studies also provide a cone-only platform to design cone-targeted therapeutic strategies for X-linked RP2.

Developing suitable medicines for genetic diseases requires a detailed understanding of not only the pathways that cause the disease, but also the identification of the genetic components involved in disease manifestation. This article focuses on the complexities associated with ocular ciliopathies – a class of debilitating disorders of the eye caused by ciliary dysfunction. Ciliated cell types have been identified in both the anterior and posterior segments of the eye. Photoreceptors (rods and cones) are the most studied ciliated neurons in the retina, which is located in the posterior eye. The photoreceptors contain a specialized lightsensing outer segment, or cilium. Any defects in the development or maintenance of the outer segment can result in severe retinal ciliopathies, such as retinitis pigmentosa and Leber congenital amaurosis. A role of cilia in the cell types involved in regulating aqueous fluid outflow in the anterior segment of the eye has also been recognized. Defects in these cell types are frequently associated with some forms of glaucoma. Here, we will discuss the significance of understanding the genetic heterogeneity and the pathogenesis of ocular ciliopathies to develop suitable treatment strategies for these blinding disorders.

This article summarizes the recent advances in our understanding of a major retinal disease gene RPGR (retinitis pigmentosa GTPase regulator), mutations in which are associated with majority of X-linked forms of retinal degenerations. A great deal of work has been done to uncover the ciliary localization of RPGR and its interacting proteins in the retina. However, the molecular mechanisms of action of RPGR in the photoreceptors are still unclear. Recent studies have begun to shed light on the intracellular pathways in which RPGR is likely involved. The deregulation of such pathways may underlie the pathogenesis of severe retinal degeneration associated with RPGR. With the recent advances in the gene augmentation therapy for RPGR-associated disease, there is a lot of excitement in the field. Patients with RPGR mutations, however, present with clinically heterogeneous manifestations. It is therefore imperative to examine the function of RPGR in detail, so that we can design patient-oriented therapeutic strategies for this disease.

Mutations in the cilia-centrosomal protein CEP290 are frequently observed in autosomal recessive childhood blindness disorder Leber congenital amaurosis (LCA). No treatment or cure currently exists for this disorder. The Cep290 ^rd16 (retinal degeneration 16) mouse (a model of LCA) carries a mutation in the Cep290 gene. This mutation leads to shorter cilia formation and defective photoreceptor structure and function. A roadblock to developing a gene replacement strategy for CEP290 using conventional adeno-associated virus (AAV) vectors is its large size. The identification and characterization is reported of a miniCEP290 gene that is amenable to AAV2/8-mediated delivery and delaying retinal degeneration in the Cep290 ^rd16 mice. Using the ability of Cep290 ^rd16 mouse embryonic fibroblasts to from shorter cilia as a platform, a human CEP290 domain encoded by amino acids 580–1180 (miniCEP290 ^580-1180 ) was identified that can recover the cilia length in vitro . Furthermore, subretinal injection of AAV particles carrying the cDNA expressing miniCEP290 ^580-1180 into neonatal Cep290 ^rd16 mice resulted in significantly improved photoreceptor survival, morphology, and function compared to control injected mice. These studies show the potential of using a truncated CEP290 to treat this fast progressing and devastating disease.

Ciliopathies are a group of severe developmental disorders that are clinically and genetically heterogeneous. They occur due to the dysfunction of cilia, which are microtubule-based extensions of the plasma membrane. The ciliary membrane acts as a hub for transmembrane receptors and specific lipids and proteins that assist in the detection and downstream signaling of extrinsic cues. They carry out diverse signaling cascades, such as sonic hedgehog signaling and sensory perceptions, including olfaction, chemosensation and photoreception (1,2). Not surprisingly, ciliary dysfunction results in severe developmental and systemic disorders, including Meckel-Gruber Syndrome, Joubert Syndrome, Bardet-Biedl Syndrome, and Senior-Loken Syndrome (1).

Ciliary trafficking defects underlie the pathogenesis of severe human ciliopathies, including Joubert Syndrome (JBTS), Bardet-Biedl Syndrome, and some forms of retinitis pigmentosa (RP). Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are common causes of RP-associated photoreceptor degeneration worldwide. While previous work has suggested that the localization of RPGR to cilia is critical to its functions, the mechanism by which RPGR and its associated cargo are trafficked to the cilia is unclear. Using proteomic and biochemical approaches, we show that RPGR interacts with two JBTS-associated ciliary proteins: PDE6δ (delta subunit of phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E). We find that PDE6δ binds selectively to the C-terminus of RPGR and that this interaction is critical for RPGR’s localization to cilia. Furthermore, we show that INPP5E associates with the N-terminus of RPGR and trafficking of INPP5E to cilia is dependent upon the ciliary localization of RPGR. These results implicate prenylation of RPGR as a critical modification for its localization to cilia and, in turn suggest that trafficking of INPP5E to cilia depends upon the interaction of RPGR with PDE6δ. Finally, our results implicate INPP5E, a novel RPGR-interacting protein, in the pathogenesis of RPGR-associated ciliopathies.