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Conde P, Rodriguez M, van der Touw W, Jimenez A, Burns M, Miller J, Brahmachary M, Chen H-M, Boros P, Rausell-Palamos F, Yun TJ, Riquelme P, Rastrojo A, Aguado B, Stein-Streilein J, Tanaka M, Zhou L, Zhang J, Lowary TL, Ginhoux F, Park CG, Cheong C, Brody J, Turley SJ, Lira SA, Bronte V, Gordon S, Heeger PS, Merad M, Hutchinson J, Chen S-H, Ochando J. DC-SIGN(+) Macrophages Control the Induction of Transplantation Tolerance. Immunity 2015;42(6):1143-58.Abstract

Tissue effector cells of the monocyte lineage can differentiate into different cell types with specific cell function depending on their environment. The phenotype, developmental requirements, and functional mechanisms of immune protective macrophages that mediate the induction of transplantation tolerance remain elusive. Here, we demonstrate that costimulatory blockade favored accumulation of DC-SIGN-expressing macrophages that inhibited CD8(+) T cell immunity and promoted CD4(+)Foxp3(+) Treg cell expansion in numbers. Mechanistically, that simultaneous DC-SIGN engagement by fucosylated ligands and TLR4 signaling was required for production of immunoregulatory IL-10 associated with prolonged allograft survival. Deletion of DC-SIGN-expressing macrophages in vivo, interfering with their CSF1-dependent development, or preventing the DC-SIGN signaling pathway abrogated tolerance. Together, the results provide new insights into the tolerogenic effects of costimulatory blockade and identify DC-SIGN(+) suppressive macrophages as crucial mediators of immunological tolerance with the concomitant therapeutic implications in the clinic.

Cruz NM, Yuan Y, Leehy BD, Baid R, Kompella U, Deangelis MM, Escher P, Haider NB. Modifier genes as therapeutics: the nuclear hormone receptor Rev Erb alpha (Nr1d1) rescues Nr2e3 associated retinal disease. PLoS One 2014;9(1):e87942.Abstract
Nuclear hormone receptors play a major role in many important biological processes. Most nuclear hormone receptors are ubiquitously expressed and regulate processes such as metabolism, circadian function, and development. They function in these processes to maintain homeostasis through modulation of transcriptional gene networks. In this study we evaluate the effectiveness of a nuclear hormone receptor gene to modulate retinal degeneration and restore the integrity of the retina. Currently, there are no effective treatment options for retinal degenerative diseases leading to progressive and irreversible blindness. In this study we demonstrate that the nuclear hormone receptor gene Nr1d1 (Rev-Erbα) rescues Nr2e3-associated retinal degeneration in the rd7 mouse, which lacks a functional Nr2e3 gene. Mutations in human NR2E3 are associated with several retinal degenerations including enhanced S cone syndrome and retinitis pigmentosa. The rd7 mouse, lacking Nr2e3, exhibits an increase in S cones and slow, progressive retinal degeneration. A traditional genetic mapping approach previously identified candidate modifier loci. Here, we demonstrate that in vivo delivery of the candidate modifier gene, Nr1d1 rescues Nr2e3 associated retinal degeneration. We observed clinical, histological, functional, and molecular restoration of the rd7 retina. Furthermore, we demonstrate that the mechanism of rescue at the molecular and functional level is through the re-regulation of key genes within the Nr2e3-directed transcriptional network. Together, these findings reveal the potency of nuclear receptors as modulators of disease and specifically of NR1D1 as a novel therapeutic for retinal degenerations.
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Di Gioia SA, Shaaban S, Tüysüz B, Elcioglu NH, Chan W-M, Robson CD, Ecklund K, Gilette NM, Hamzaoglu A, Tayfun GA, Traboulsi EI, Engle EC. Recessive MYF5 Mutations Cause External Ophthalmoplegia, Rib, and Vertebral Anomalies. Am J Hum Genet 2018;103(1):115-124.Abstract
MYF5 is member of the Myc-like basic helix-loop-helix transcription factor family and, in cooperation with other myogenic regulatory factors MYOD and MYF5, is a key regulator of early stages of myogenesis. Here, we report three consanguineous families with biallelic homozygous loss-of-function mutations in MYF5 who define a clinical disorder characterized by congenital ophthalmoplegia with scoliosis and vertebral and rib anomalies. The clinical phenotype overlaps strikingly with that reported in several Myf5 knockout mouse models. Affected members of two families share a haploidentical region that contains a homozygous 10 bp frameshift mutation in exon 1 of MYF5 (c.23_32delAGTTCTCACC [p.Gln8Leufs86]) predicted to undergo nonsense-mediated decay. Affected members of the third family harbor a homozygous missense change in exon 1 of MYF5 (c.283C>T [p.Arg95Cys]). Using in vitro assays, we show that this missense mutation acts as a loss-of-function allele by impairing MYF5 DNA binding and nuclear localization. We performed whole-genome sequencing in one affected individual with the frameshift mutation and did not identify additional rare variants in the haploidentical region that might account for differences in severity among the families. These data support the direct role of MYF5 in rib, spine, and extraocular muscle formation in humans.
Drokhlyansky E, Soh TK, Cepko CL. Preferential Budding of Vesicular Stomatitis Virus from the Basolateral Surface of Polarized Epithelial Cells Is Not Solely Directed by Matrix Protein or Glycoprotein. J Virol 2015;89(22):11718-22.Abstract

Vesicular stomatitis virus has been shown to bud basolaterally, and the matrix protein, but not glycoprotein, was proposed to mediate this asymmetry. Using polarized T84 monolayers, we demonstrate that no single viral protein is sufficient for polarized budding. Particles are released from the apical and basolateral surfaces and are indistinguishable, indicating that there is no apical assembly defect. We propose that aspects of host cell polarity create a more efficient budding process at the basolateral surface.

Dudek AM, Pillay S, Puschnik AS, Nagamine CM, Cheng F, Qiu J, Carette JE, Vandenberghe LH. An Alternate Route for Adeno-associated Virus (AAV) Entry Independent of AAV Receptor. J Virol 2018;92(7)Abstract
Determinants and mechanisms of cell attachment and entry steer adeno-associated virus (AAV) in its utility as a gene therapy vector. Thus far, a systematic assessment of how diverse AAV serotypes engage their proteinaceous receptor AAVR (KIAA0319L) to establish transduction has been lacking, despite potential implications for cell and tissue tropism. Here, a large set of human and simian AAVs as well as -reconstructed ancestral AAV capsids were interrogated for AAVR usage. We identified a distinct AAV capsid lineage comprised of AAV4 and AAVrh32.33 that can bind and transduce cells in the absence of AAVR, independent of the multiplicity of infection. Virus overlay assays and rescue experiments in nonpermissive cells demonstrate that these AAVs are unable to bind to or use the AAVR protein for entry. Further evidence for a distinct entry pathway was observed , as AAVR knockout mice were equally as permissive to transduction by AAVrh32.33 as wild-type mice upon systemic injection. We interestingly observe that some AAV capsids undergo a low level of transduction in the absence of AAVR, both and , suggesting that some capsids may have a multimodal entry pathway. In aggregate, our results demonstrate that AAVR usage is conserved among all primate AAVs except for those of the AAV4 lineage, and a non-AAVR pathway may be available to other serotypes. This work furthers our understanding of the entry of AAV, a vector system of broad utility in gene therapy. Adeno-associated virus (AAV) is a nonpathogenic virus that is used as a vehicle for gene delivery. Here, we have identified several situations in which transduction is retained in both cell lines and a mouse model in the absence of a previously defined entry receptor, AAVR. Defining the molecular determinants of the infectious pathway of this highly relevant viral vector system can help refine future applications and therapies with this vector.
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Ehrenberg M, Bagdonite-Bejarano L, Fulton AB, Orenstein N, Yahalom C. Genetic causes of nystagmus, foveal hypoplasia and subnormal visual acuity- other than albinism. Ophthalmic Genet 2021;42(3):243-251.Abstract
Background: To describe genetic molecular findings in individuals with congenital nystagmus, foveal hypoplasia, and subnormal vision, with normal ocular pigmentation (absence of diffuse transillumination or transparent retinal pigment typical for albinism).Methods: This is a retrospective, multicenter study of ophthalmic, systemic, and genetic features, as collected from medical records of patients diagnosed with infantile nystagmus and foveal hypoplasia. Ophthalmic findings include best-corrected visual acuity (BCVA), biomicroscopic examination, cycloplegic refraction, retinal examination, macular optical coherence tomography, and electroretinography. Genetic information was retrieved from the participating genetic clinics and included ethnicity and molecular diagnosis.Results: Thirty-one individuals met the inclusion criteria and had a secure molecular diagnosis. Mutations in two genes predominated, constituting 77.4% of all the represented genes: SLC38A8 (45.1%) and PAX6 (32.3%). Seventy-eight percent of the subjects who had a measurable BCVA had moderate and severe visual impairment (range 20/80 to 20/270). Most patients with a mutation in SLC38A8 had mild to moderate astigmatism, while most patients with PAX6 mutation had moderate and severe myopia. Patients in the PAX6 group had variable degrees of anterior segment manifestations.Conclusion: In our cohort, the main causative genes for congenital nystagmus and foveal hypoplasia in normally pigmented eyes were SLC38A8 and PAX6. A mild phenotype in PAX6 mutations may be an under-diagnosed cause of nystagmus and foveal hypoplasia. Reaching an accurate genetic diagnosis is essential for both the patients and their family members. This enables predicting disease prognosis, tailoring correct follow-up, and providing genetic counseling and family planning to affected families.
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Ferenchak K, Deitch I, Huckfeldt R. Antisense Oligonucleotide Therapy for Ophthalmic Conditions. Semin Ophthalmol 2021;36(5-6):452-457.Abstract
Antisense oligonucleotides (AON) are synthetic single-stranded fragments of nucleic acids that bind to a specific complementary messenger RNA (mRNA) sequence and change the final gene product. AON were initially approved for treating cytomegalovirus retinitis and have shown promise in treating Mendelian systemic disease. AON are currently being investigated as a treatment modality for many ophthalmic diseases, including inherited retinal disorders (IRD), inflammatory response and wound healing after glaucoma surgery, and macular degeneration. They provide a possible solution to gene therapy for IRD that are not candidates for adeno-associated virus (AAV) delivery. This chapter outlines the historical background of AON and reviews clinical applications and ongoing clinical trials.
Florea M, Nicolaou F, Pacouret S, Zinn EM, Sanmiguel J, Andres-Mateos E, Unzu C, Wagers AJ, Vandenberghe LH. High-efficiency purification of divergent AAV serotypes using AAVX affinity chromatography. Mol Ther Methods Clin Dev 2023;28:146-159.Abstract
The adeno-associated viral vector (AAV) provides a safe and efficient gene therapy platform with several approved products that have marked therapeutic impact for patients. However, a major bottleneck in the development and commercialization of AAV remains the efficiency, cost, and scalability of AAV production. Chromatographic methods have the potential to allow purification at increased scales and lower cost but often require optimization specific to each serotype. Here, we demonstrate that the POROS CaptureSelect AAVX affinity resin efficiently captures a panel of 15 divergent AAV serotypes, including the commonly used AAV2, AAV8, AAV9, PHP.B, and Anc80. We also find that AAVX resin can be regenerated repeatedly without loss of efficiency or carry-over contamination. While AAV preps purified with AAVX showed a higher fraction of empty capsids than preps purified using iodixanol ultracentrifugation, the potency of the AAVX purified vectors was comparable with that of iodixanol purified vectors both in vitro and in vivo. Finally, optimization of the purification protocol resulted in a process with an overall efficiency of 65%-80% across all scales and AAV serotypes tested. These data establish AAVX affinity chromatography as a versatile and efficient method for purification of a broad range of AAV serotypes.
Fuentealba LC, Rompani SB, Parraguez JI, Obernier K, Romero R, Cepko CL, Alvarez-Buylla A. Embryonic Origin of Postnatal Neural Stem Cells. Cell 2015;161(7):1644-55.Abstract

Adult neural stem/progenitor (B1) cells within the walls of the lateral ventricles generate different types of neurons for the olfactory bulb (OB). The location of B1 cells determines the types of OB neurons they generate. Here we show that the majority of mouse B1 cell precursors are produced between embryonic days (E) 13.5 and 15.5 and remain largely quiescent until they become reactivated postnatally. Using a retroviral library carrying over 100,000 genetic tags, we found that B1 cells share a common progenitor with embryonic cells of the cortex, striatum, and septum, but this lineage relationship is lost before E15.5. The regional specification of B1 cells is evident as early as E11.5 and is spatially linked to the production of neurons that populate different areas of the forebrain. This study reveals an early embryonic regional specification of postnatal neural stem cells and the lineage relationship between them and embryonic progenitor cells.

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Garanto A, Chung DC, Duijkers L, Corral-Serrano JC, Messchaert M, Xiao R, Bennett J, Vandenberghe LH, Collin RWJ. In vitro and in vivo rescue of aberrant splicing in CEP290-associated LCA by antisense oligonucleotide delivery. Hum Mol Genet 2016;Abstract

Leber congenital amaurosis (LCA) is a severe disorder resulting in visual impairment usually starting in the first year of life. The most frequent genetic cause of LCA is an intronic mutation in CEP290 (c.2991+1655A>G) that creates a cryptic splice donor site resulting in the insertion of a pseudoexon (exon X) into CEP290 mRNA. Previously, we showed that naked antisense oligonucleotides (AONs) effectively restored normal CEP290 splicing in patient-derived lymphoblastoid cells. We here explore the therapeutic potential treatment of naked and AAV-packaged AONs in vitro and in vivo In both cases, AON delivery fully restored CEP290 pre-mRNA splicing, significantly increased CEP290 protein levels and rescued a ciliary phenotype present in patient-derived fibroblast cells. Moreover, administration of naked and AAV-packaged AONs to the retina of a humanized mutant Cep290 mouse model, carrying the intronic mutation, showed a statistically significant reduction of exon X-containing Cep290 transcripts, without compromising the retinal structure. Together, our data highlight the tremendous therapeutic prospective of AONs for the treatment of not only CEP290-associated LCA but potentially many other subtypes of retinal dystrophy caused by splicing mutations.

Girach A, Audo I, Birch DG, Huckfeldt RM, Lam BL, Leroy BP, Michaelides M, Russell SR, Sallum JMF, Stingl K, Tsang SH, Yang P. RNA-based therapies in inherited retinal diseases. Ther Adv Ophthalmol 2022;14:25158414221134602.Abstract
Inherited retinal diseases (IRDs) are a genetically and phenotypically heterogeneous group of genetic eye disorders. There are more than 300 disease entities, and together this group of disorders affects millions of people globally and is a frequent cause of blindness or low-vision certification. However, each type is rare or ultra-rare. Characteristically, the impaired vision in IRDs is due to retinal photoreceptor dysfunction and loss resulting from mutation in a gene that codes for a retinal protein. Historically, IRDs have been considered incurable and individuals living with these blinding conditions could be offered only supportive care. However, the treatment landscape for IRDs is beginning to evolve. Progress is being made, driven by improvements in understanding of genotype-phenotype relationships, through advances in molecular genetic testing and retinal imaging. Alongside this expanding knowledge of IRDs, the current era of precision medicine is fueling a growth in targeted therapies. This has resulted in the first treatment for an IRD being approved. Several other therapies are currently in development in the IRD space, including RNA-based therapies, gene-based therapies (such as augmentation therapy and gene editing), cell therapy, visual prosthetics, and optogenetics. RNA-based therapies are a novel approach within precision medicine that have demonstrated success, particularly in rare diseases. Three antisense oligonucleotides (AONs) are currently in development for the treatment of specific IRD subtypes. These RNA-based therapies bring several key advantages in the setting of IRDs, and the potential to bring meaningful vision benefit to individuals living with inherited blinding disorders. This review will examine the increasing breadth and relevance of RNA-based therapies in clinical medicine, explore the key features that make AONs suitable for treating genetic eye diseases, and provide an overview of the three-leading investigational AONs in clinical trials.
Grotz S, Schäfer J, Wunderlich KA, Ellederova Z, Auch H, Bähr A, Runa-Vochozkova P, Fadl J, Arnold V, Ardan T, Veith M, Santamaria G, Dhom G, Hitzl W, Kessler B, Eckardt C, Klein J, Brymova A, Linnert J, Kurome M, Zakharchenko V, Fischer A, Blutke A, Döring A, Suchankova S, Popelar J, Rodríguez-Bocanegra E, Dlugaiczyk J, Straka H, May-Simera H, Wang W, Laugwitz K-L, Vandenberghe LH, Wolf E, Nagel-Wolfrum K, Peters T, Motlik J, Fischer DM, Wolfrum U, Klymiuk N. Early disruption of photoreceptor cell architecture and loss of vision in a humanized pig model of usher syndromes. EMBO Mol Med 2022;14(4):e14817.Abstract
Usher syndrome (USH) is the most common form of monogenic deaf-blindness. Loss of vision is untreatable and there are no suitable animal models for testing therapeutic strategies of the ocular constituent of USH, so far. By introducing a human mutation into the harmonin-encoding USH1C gene in pigs, we generated the first translational animal model for USH type 1 with characteristic hearing defect, vestibular dysfunction, and visual impairment. Changes in photoreceptor architecture, quantitative motion analysis, and electroretinography were characteristics of the reduced retinal virtue in USH1C pigs. Fibroblasts from USH1C pigs or USH1C patients showed significantly elongated primary cilia, confirming USH as a true and general ciliopathy. Primary cells also proved their capacity for assessing the therapeutic potential of CRISPR/Cas-mediated gene repair or gene therapy in vitro. AAV-based delivery of harmonin into the eye of USH1C pigs indicated therapeutic efficacy in vivo.
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Hafler BP. CLINICAL PROGRESS IN INHERITED RETINAL DEGENERATIONS: GENE THERAPY CLINICAL TRIALS AND ADVANCES IN GENETIC SEQUENCING. Retina 2017;37(3):417-423.Abstract

PURPOSE: Inherited retinal dystrophies are a significant cause of vision loss and are characterized by the loss of photoreceptors and the retinal pigment epithelium (RPE). Mutations in approximately 250 genes cause inherited retinal degenerations with a high degree of genetic heterogeneity. New techniques in next-generation sequencing are allowing the comprehensive analysis of all retinal disease genes thus changing the approach to the molecular diagnosis of inherited retinal dystrophies. This review serves to analyze clinical progress in genetic diagnostic testing and implications for retinal gene therapy. METHODS: A literature search of PubMed and OMIM was conducted to relevant articles in inherited retinal dystrophies. RESULTS: Next-generation genetic sequencing allows the simultaneous analysis of all the approximately 250 genes that cause inherited retinal dystrophies. Reported diagnostic rates range are high and range from 51% to 57%. These new sequencing tools are highly accurate with sensitivities of 97.9% and specificities of 100%. Retinal gene therapy clinical trials are underway for multiple genes including RPE65, ABCA4, CHM, RS1, MYO7A, CNGA3, CNGB3, ND4, and MERTK for which a molecular diagnosis may be beneficial for patients. CONCLUSION: Comprehensive next-generation genetic sequencing of all retinal dystrophy genes is changing the paradigm for how retinal specialists perform genetic testing for inherited retinal degenerations. Not only are high diagnostic yields obtained, but mutations in genes with novel clinical phenotypes are also identified. In the era of retinal gene therapy clinical trials, identifying specific genetic defects will increasingly be of use to identify patients who may enroll in clinical studies and benefit from novel therapies.

Harwell CC, Fuentealba LC, Gonzalez-Cerrillo A, Parker PRL, Gertz CC, Mazzola E, Garcia MT, Alvarez-Buylla A, Cepko CL, Kriegstein AR. Wide Dispersion and Diversity of Clonally Related Inhibitory Interneurons. Neuron 2015;87(5):999-1007.Abstract

The mammalian neocortex is composed of two major neuronal cell types with distinct origins: excitatory pyramidal neurons and inhibitory interneurons, generated in dorsal and ventral progenitor zones of the embryonic telencephalon, respectively. Thus, inhibitory neurons migrate relatively long distances to reach their destination in the developing forebrain. The role of lineage in the organization and circuitry of interneurons is still not well understood. Utilizing a combination of genetics, retroviral fate mapping, and lineage-specific retroviral barcode labeling, we find that clonally related interneurons can be widely dispersed while unrelated interneurons can be closely clustered. These data suggest that migratory mechanisms related to the clustering of interneurons occur largely independent of their clonal origin.

Heidary G, Calderwood L, Cox GF, Robson CD, Teot LA, Mullon J, Anselm I. Optic atrophy and a Leigh-like syndrome due to mutations in the c12orf65 gene: report of a novel mutation and review of the literature. J Neuroophthalmol 2014;34(1):39-43.Abstract
Combined oxidative phosphorylation deficiency type 7 (COXPD7) is a rare disorder of mitochondrial metabolism that results in optic atrophy and Leigh syndrome-like disease. We describe 2 siblings with compound heterozygous mutations in the recently identified C12orf65 gene who presented with optic atrophy and mild developmental delays and subsequently developed bilateral, symmetric lesions in the brainstem reminiscent of Leigh syndrome. Repeat neuroimaging demonstrated reversibility of the findings in 1 sibling and persistent metabolic stroke in the other. This article highlights the phenotypic manifestations from a novel mutation in the C12orf65 gene and reviews the clinical presentation of the 5 other individuals reported to date who carry mutations in this gene.
Hildebrandt C, Fulton A, Rodan LH. Homozygous deletion of 21q22.2 in a patient with hypotonia, developmental delay, cortical visual impairment, and retinopathy. Am J Med Genet A 2021;185(2):555-560.Abstract
21q22 contains several dosage sensitive genes that are important in neurocognitive development. Determining impacts of gene dosage alterations in this region can be useful in establishing contributions of these genes to human development and disease. We describe a 15-month-old girl with a 1,140 kb homozygous deletion in the Down Syndrome Critical Region at 21q22.2 including 4 genes; B3GALT5, IGSF5, PCP4, DSCAM, and a microRNA (MIR4760). Clinical singleton genome sequencing did not report any candidate gene variants for the patient's phenotype. She presented with hypotonia, global developmental delay, cortical visual impairment, and mild facial dysmorphism. Ophthalmological exam was suggestive of retinopathy. We propose that the absence of DSCAM and PCP4 may contribute to the patient's neurological and retinal phenotype, while the role of absent B3GALT5 and IGSF5 in her presentation remain unclear at this time.
Hou Y, Lin H, Zhu L, Liu Z, Hu F, Shi J, Yang T, Shi X, Guo H, Tan X, Zhang L, Wang Q, Li Z, Zhao Y. The inhibitory effect of IFN-γ on protease HTRA1 expression in rheumatoid arthritis. J Immunol 2014;193(1):130-8.Abstract
The high temperature requirement A1 (HTRA1) is a potent protease involved in many diseases, including rheumatoid arthritis (RA). However, the regulatory mechanisms that control HTRA1 expression need to be determined. In this study, we demonstrated that IFN-γ significantly inhibited the basal and LPS-induced HTRA1 expression in fibroblasts and macrophages, which are two major cells for HTRA1 production in RA. Importantly, the inhibitory effect of IFN-γ on HTRA1 expression was evidenced in collagen-induced arthritis (CIA) mouse models and in human RA synovial cells. In parallel with the enhanced CIA incidence and pathological changes in IFN-γ-deficient mice, HTRA1 expression in the joint tissues was also increased as determined by real-time PCR and Western blots. IFN-γ deficiency increased the incidence of CIA and the pathological severity in mice. Neutralization of HTRA1 by Ab significantly reversed the enhanced CIA frequency and severity in IFN-γ-deficient mice. Mechanistically, IFN-γ negatively controls HTRA1 expression through activation of p38 MAPK/STAT1 pathway. Dual luciferase reporter assay and chromatin immunoprecipitation analysis showed that STAT1 could directly bind to HTRA1 promoter after IFN-γ stimulation. This study offers new insights into the molecular regulation of HTRA1 expression and its role in RA pathogenesis, which may have significant impact on clinical therapy for RA and possibly other HTRA1-related diseases, including osteoarthritis, age-related macular degeneration, and cancer.
Hsu Y, Bhattarai S, Thompson JM, Mahoney A, Thomas J, Mayer SK, Datta P, Garrison J, Searby CC, Vandenberghe LH, Seo S, Sheffield VC, Drack AV. Subretinal gene therapy delays vision loss in a Bardet-Biedl Syndrome type 10 mouse model. Mol Ther Nucleic Acids 2023;31:164-181.Abstract
Blindness in Bardet-Biedl syndrome (BBS) is caused by dysfunction and loss of photoreceptor cells in the retina. BBS10, mutations of which account for approximately 21% of all BBS cases, encodes a chaperonin protein indispensable for the assembly of the BBSome, a cargo adaptor important for ciliary trafficking. The loss of BBSome function in the eye causes a reduced light sensitivity of photoreceptor cells, photoreceptor ciliary malformation, dysfunctional ciliary trafficking, and photoreceptor cell death. Cone photoreceptors lacking BBS10 have congenitally low electrical function in electroretinography. In this study, we performed gene augmentation therapy by injecting a viral construct subretinally to deliver the coding sequence of the mouse Bbs10 gene to treat retinal degeneration in a BBS10 mouse model. Long-term efficacy was assessed by measuring the electrical functions of the retina over time, imaging of the treated regions to visualize cell survival, conducting visually guided swim assays to measure functional vision, and performing retinal histology. We show that subretinal gene therapy slowed photoreceptor cell death and preserved retinal function in treated eyes. Notably, cone photoreceptors regained their electrical function after gene augmentation. Measurement of functional vision showed that subretinal gene therapy provided a significant benefit in delaying vision loss.
Hu Z, Cano I, Saez-Torres KL, LeBlanc ME, Saint-Geniez M, Ng Y-S, Argüeso P, D'Amore PA. Elements of the Endomucin Extracellular Domain Essential for VEGF-Induced VEGFR2 Activity. Cells 2020;9(6)Abstract
Endomucin (EMCN) is the type I transmembrane glycoprotein, mucin-like component of the endothelial cell glycocalyx. We have previously shown that EMCN is necessary for vascular endothelial growth factor (VEGF)-induced VEGF receptor 2 (VEGFR2) internalization and downstream signaling. To explore the structural components of EMCN that are necessary for its function and the molecular mechanism of EMCN in VEGF-induced endothelial functions, we generated a series of mouse EMCN truncation mutants and examined their ability to rescue VEGF-induced endothelial functions in human primary endothelial cells (EC) in which endogenous EMCN had been knocked down using siRNA. Expression of the mouse full-length EMCN (FL EMCN) and the extracellular domain truncation mutants ∆21-81 EMCN and ∆21-121 EMCN, but not the shortest mutant ∆21-161 EMCN, successfully rescued the VEGF-induced EC migration, tube formation, and proliferation. ∆21-161 EMCN failed to interact with VEGFR2 and did not facilitate VEGFR2 internalization. Deletion of COSMC (C1GalT1C1) revealed that the abundant mucin-type -glycans were not required for its VEGFR2-related functions. Mutation of the two -glycosylation sites on ∆21-121 EMCN abolished its interaction with VEGFR2 and its function in VEGFR2 internalization. These results reveal ∆21-121 EMCN as the minimal extracellular domain sufficient for VEGFR2-mediated endothelial function and demonstrate an important role for -glycosylation in VEGFR2 interaction, internalization, and angiogenic activity.
Huckfeldt R, Sobrin L. BEST1-One Gene, Many Diseases. JAMA Ophthalmol 2020;

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