The Road to Ocular Gene Therapy: Lessons from the Past

This presentation at the Eyes to the Past exhibit reception by Hemant Khanna, PhD, principal investigator and associate professor, Department of Ophthalmology and Visual Sciences, UMass Medical School, discusses the learnings gathered from ocular gene therapy over the years and new discoveries taking place at UMass Medical School.https://escholarship.umassmed.edu/eyes-to-the-past-presentations/1000/thumbnail.jp

The Ocular Gene Delivery Landscape

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

Advances in Gene Therapy for Diseases of the Eye

Over the last few years, huge progress has been made with regard to the understanding of molecular mechanisms underlying the pathogenesis of neurodegenerative diseases of the eye. Such knowledge has led to the development of gene therapy approaches to treat these devastating disorders. Challenges regarding the efficacy and efficiency of therapeutic gene delivery have driven the development of novel therapeutic approaches, which continue to evolve the field of ocular gene therapy. In this review article, we will discuss the evolution of preclinical and clinical strategies that have improved gene therapy in the eye, showing that treatment of vision loss has a bright future

The carboxyl terminal mutational hotspot of the ciliary disease protein RPGRORF15 (retinitis pigmentosa GTPase regulator) is glutamylated in vivo

Mutations inRPGR(ORF15)(retinitis pigmentosa GTPase regulator) are a major cause of inherited retinal degenerative diseases. RPGR(ORF15)(1152 residues) is a ciliary protein involved in regulating the composition and function of photoreceptor cilia. The mutational hotspot in RPGR(ORF15)is an unusual C-terminal domain encoded by exon ORF15, which is rich in polyglutamates and glycine residues (Glu-Gly domain) followed by a short stretch of basic amino acid residues (RPGR(C2)domain; residues 1072-1152). However, the properties of the ORF15-encoded domain and its involvement in the pathogenesis of the disease are unclear. Here we show that RPGR(ORF15)is glutamylated at the C-terminus, as determined by binding to GT335, which recognizes glutamylated substrates. This reactivity is lost in two mouse mutants ofRpgr, which do not express RPGR(ORF15)due to disease-causing mutations in exon ORF15. Our results indicate that RPGR(ORF15)is posttranslationally glutamylated in the Glu-Gly domain and that the GT335 antibody predominantly recognizes RPGR(ORF15)in photoreceptor cilia

Structural but Not Functional Alterations in Cones in the Absence of the Retinal Disease Protein Retinitis Pigmentosa 2 (RP2) in a Cone-Only Retina

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

Ablation of retinal ciliopathy protein RPGR results in altered photoreceptor ciliary composition

Cilia regulate several developmental and homeostatic pathways that are critical to survival. Sensory cilia of photoreceptors regulate phototransduction cascade for visual processing. Mutations in the ciliary protein RPGR (retinitis pigmentosa GTPase regulator) are a prominent cause of severe blindness disorders due to degeneration of mature photoreceptors. However, precise function of RPGR is still unclear. Here we studied the involvement of RPGR in ciliary trafficking by analyzing the composition of photoreceptor sensory cilia (PSC) in Rpgr(ko) retina. Using tandem mass spectrometry analysis followed by immunoblotting, we detected few alterations in levels of proteins involved in proteasomal function and vesicular trafficking in Rpgr(ko) PSC, prior to onset of degeneration. We also found alterations in the levels of high molecular weight soluble proteins in Rpgr(ko) PSC. Our data indicate RPGR regulates entry or retention of soluble proteins in photoreceptor cilia but spares the trafficking of key structural and phototransduction-associated proteins. Given a frequent occurrence of RPGR mutations in severe photoreceptor degeneration due to ciliary disorders, our results provide insights into pathways resulting in altered mature cilia function in ciliopathies

Gene Therapy Using a miniCEP290 Fragment Delays Photoreceptor Degeneration in a Mouse Model of Leber Congenital Amaurosis

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

Prenylated retinal ciliopathy protein RPGR regulates ciliary localization of Joubert Syndrome-associated protein INPP5E in cooperation with PDE6

Ciliary dysfunction is an underlying cause of severe human disorders (collectively called ciliopathies), such as retinitis pigmentosa (RP), Joubert Syndrome (JBTS), and Bardet-Biedl Syndrome. Ciliary proteins form distinct functional networks for localization to cilia as well as regulation of ciliary function. However, not much is known about the mechanism of ciliary localization and function of RPGR (retinitis pigmentosa GTPase regulator), a ciliary protein frequently associated with RP worldwide. Using tandem mass spectrometry analysis, we show that RPGR interacts with two JBTS-associated proteins: PDE6Π (delta subunit of Phosphodiesterase; a prenyl-binding protein) and INPP5E (inositol polyphosphate-5-phosphatase 5E; a ciliary cargo). Whereas PDE6Π binds in a prenylation-dependent manner to the C-terminus of RPGR, INPP5E associates with the N-terminus of RPGR. Prenylation and interaction of RPGR with PDE6Π are critical for its localization to cilia. We further show that loss of RPGR results in reduced amount of INPP5E in cilia of fibroblasts and in photoreceptor outer segment, a modified sensory cilium. Overall, our results suggest that RPGR, in complex with PDE6D, regulates the trafficking of ciliary cargo INPP5E and implicate reduction in ciliary INPP5E in the pathogenesis of RPGR-ciliopathy