Background: In retinitis pigmentosa (RP) rod photoreceptors degenerate from one of many mutations after which cones are compromised by oxidative stress. N-acetylcysteine (NAC) reduces oxidative damage and increases cone function/survival in RP models. We tested the safety, tolerability, and visual function effects of oral NAC in RP patients. Methods: Subjects (n = 10 per cohort) received 600 mg (cohort 1), 1200 mg (cohort 2), or 1800 mg (cohort 3) NAC BID for 12 weeks and then TID for 12 weeks. Best-corrected visual acuity (BCVA), macular sensitivity, ellipsoid zone (EZ) width, and aqueous NAC were measured. Linear mixed effects models were used to estimate the rates of changes during the treatment period. Results: There were 9 drug-related gastrointestinal adverse events which resolved spontaneously or with dose reduction (MTD 1800 mg bid). During the 24 week treatment period, mean BCVA significantly improved at 0.4 (95% CI 0.2–0.6, P < 0.001), 0.5 (95% CI 0.3–0.7, P < 0.001) and 0.2 (95% CI 0.02–0.4, P = 0.03) letters/month in cohorts 1, 2 and 3, respectively. There was no significant improvement in mean sensitivity (MS) over time in cohorts 1 and 2, but there was in cohort 3 (0.15 dB/month, 95%CI 0.04–0.26). There was no significant change in mean EZ width in any cohort. Conclusion: Oral NAC is safe and well-tolerated in patients with moderately advanced RP and may improve suboptimally functioning macular cones. A randomized, placebo-controlled trial is needed to determine if oral NAC can provide long term stabilization and/or improvement in visual function in patients with RP.
Peter A. Campochiaro, Mustafa Iftikhar, Gulnar Hafiz, Anam Akhlaq, Grace Tsai, Dagmar Wehling, Lili Lu, G. Michael Wall, Mandeep S. Singh, Xiangrong Kong
Increases in the number of cell therapies in the preclinical and clinical phases have prompted the need for reliable and non-invasive assays to validate transplant function in clinical biomanufacturing. We developed a robust characterization methodology composed of quantitative bright-field absorbance microscopy (QBAM) and deep neural networks (DNNs) to non-invasively predict tissue function and cellular donor identity. The methodology was validated using clinical-grade induced pluripotent stem cell derived retinal pigment epithelial cells (iPSC-RPE). QBAM images of iPSC-RPE were used to train DNNs that predicted iPSC-RPE monolayer transepithelial resistance, predicted polarized vascular endothelial growth factor (VEGF) secretion, and matched iPSC-RPE monolayers to the stem cell donors. DNN predictions were supplemented with traditional machine learning algorithms that identified shape and texture features of single cells that were used to predict tissue function and iPSC donor identity. These results demonstrate non-invasive cell therapy characterization can be achieved with QBAM and machine learning.
Nicholas J. Schaub, Nathan A. Hotaling, Petre Manescu, Sarala Padi, Qin Wan, Ruchi Sharma, Aman George, Joe Chalfoun, Mylene Simon, Mohamed Ouladi, Carl G. Simon, Jr., Peter Bajcsy, Kapil Bharti
Mutations in genes encoding components of the mitochondrial DNA (mtDNA) replication machinery cause mtDNA depletion syndromes (MDS), which associate ocular features with severe neurological syndromes. Here, we identified heterozygous missense mutations in SSBP1 in five unrelated families, leading to the R38Q and R107Q amino-acid changes in the mitochondrial single-stranded DNA-binding protein, a crucial protein involved in mtDNA replication. All affected individuals presented optic atrophy, associated with foveopathy in half of the cases. To uncover the structural features underlying SSBP1 mutations, we determined a new revised SSBP1 crystal structure. Structural analysis suggests that both mutations affect dimer interactions and presumably distort the DNA binding region. Using patient fibroblasts, we validated that the R38Q variant destabilizes SSBP1 dimer/tetramer formation, affects mtDNA replication and induces mtDNA depletion. Our study, showing that mutations in SSBP1 cause a novel form of dominant optic atrophy frequently accompanied with foveopathy, brings new insights into mtDNA maintenance disorders.
Camille Piro-Mégy, Emmanuelle Sarzi, Aleix Tarrés-Solé, Marie Péquignot, Fenna Hensen, Mélanie Quilès, Gaël Manes, Arka Chakraborty, Audrey Sénéchal, Béatrice Bocquet, Chantal Cazevieille, Agathe Roubertie, Agnès Müller, Majida Charif, David Goudenège, Guy Lenaers, Helmut Wilhelm, Ulrich Kellner, Nicole Weisschuh, Bernd Wissinger, Xavier Zanlonghi, Christian Hamel, Johannes N. Spelbrink, Maria Solà, Cécile Delettre
Inherited optic neuropathies include complex phenotypes, mostly driven by mitochondrial dysfunction. We report an optic atrophy spectrum disorder, including retinal macular dystrophy and kidney insufficiency leading to transplantation, associated with mitochondrial DNA (mtDNA) depletion without accumulation of multiple deletions. By whole-exome sequencing, we identified mutations affecting the mitochondrial single strand binding protein (SSBP1) in four families with dominant and one with recessive inheritance. We show that SSBP1 mutations in patient-derived fibroblasts variably affect its amount and alter multimer formation, but not the binding to ssDNA. SSBP1 mutations impaired mtDNA, nucleoids and 7S-DNA amounts as well as mtDNA replication, impacting replisome machinery. The variable mtDNA depletion in cells reflected in severity of mitochondrial dysfunction, including respiratory efficiency, OXPHOS subunits and complexes amount and assembly. mtDNA depletion and cytochrome c oxidase-negative cells were found ex-vivo in biopsies of affected tissues, like kidney and skeletal muscle. Reduced efficiency of mtDNA replication was also reproduced in vitro, confirming the pathogenic mechanism. Furthermore, ssbp1 suppression in zebrafish induced signs of nephropathy and reduced optic nerve size, the latter phenotype complemented by wild-type mRNA but not by SSBP1 mutant transcripts. This previously unrecognized disease of mtDNA maintenance implicates SSBP1 mutations as cause of human pathology.
Valentina Del Dotto, Farid Ullah, Ivano Di Meo, Pamela Magini, Mirjana Gusic, Alessandra Maresca, Leonardo Caporali, Flavia Palombo, Francesca Tagliavini, Evan H. Baugh, Bertil Macao, Zsolt Szilagyi, Camille Péron, Margaret A. Gustafson, Kamal Khan, Chiara La Morgia, Piero Barboni, Michele Carbonelli, Maria Lucia Valentino, Rocco Liguori, Vandana Shashi, Jennifer A. Sullivan, Shashi Nagaraj, Mays El-Dairi, Alessandro Iannaccone, Ioana Cutcutache, Enrico Bertini, Rosalba Carrozzo, Francesco Emma, Francesca Diomedi-Camassei, Claudia Zanna, Martin Armstrong, Matthew J Page, Sylvia Boesch, Saskia B. Wortmann, Robert Kopajtich, Nicholas Stong, Wolfgang Sperl, Erica Davis, William C. Copeland, Marco Seri, Maria Falkenberg, Holger Prokisch, Nicholas Katsanis, Valeria Tiranti, Tommaso Pippucci, Valerio Carelli
The majority of patients with diabetic macular edema (DME), the most common cause of vision loss in working-age Americans, do not respond adequately to current therapies targeting VEGFA. Here, we show that expression of angiopoietin-like 4 (ANGPTL4), a HIF-1–regulated gene product, is increased in the eyes of diabetic mice and patients with DME. We observed that ANGPTL4 and VEGF act synergistically to destabilize the retinal vascular barrier. Interestingly, while ANGPTL4 modestly enhanced tyrosine phosphorylation of VEGF receptor 2, promotion of vascular permeability by ANGPTL4 was independent of this receptor. Instead, we found that ANGPTL4 binds directly to neuropilin 1 (NRP1) and NRP2 on endothelial cells (ECs), leading to rapid activation of the RhoA/ROCK signaling pathway and breakdown of EC-EC junctions. Treatment with a soluble fragment of NRP1 (sNRP1) prevented ANGPTL4 from binding to NRP1 and blocked ANGPTL4-induced activation of RhoA as well as EC permeability in vitro and retinal vascular leakage in diabetic animals in vivo. In addition, sNRP1 reduced the stimulation of EC permeability by aqueous fluid from patients with DME. Collectively, these data identify the ANGPTL4/NRP/RhoA pathway as a therapeutic target for the treatment of DME.
Akrit Sodhi, Tao Ma, Deepak Menon, Monika Deshpande, Kathleen Jee, Aumreetam Dinabandhu, Jordan Vancel, Daoyuan Lu, Silvia Montaner
Polyunsaturated fatty acids (PUFAs) such as docosahexaenoic acid (DHA) positively affect the outcome of retinopathy of prematurity (ROP). Given that DHA metabolism by cytochrome P450 and soluble epoxide hydrolase (sEH) enzymes affects retinal angiogenesis and vascular stability we investigated the role of sEH in a mouse model of ROP. In wild-type mice, hyperoxia elicited the tyrosine nitration and inhibition of the sEH and decreased generation of the DHA-derived diol 19,20-dihydroxydocosapentaenoic acid (DHDP). Correspondingly in a murine model of ROP, sEH–/– mice developed a larger central avascular zone and peripheral pathological vascular tuft formation than their wild-type littermates. Astrocytes were the cells most affected by sEH deletion and hyperoxia increased astrocyte apoptosis. In rescue experiments 19,20-DHDP prevented astrocyte loss by targeting the mitochondrial membrane to prevent the hyperoxia-induced dissociation of presenilin-1 (PS-1) and PS-1 associated protein (PSAP) to attenuate PARP1 activation and mitochondrial DNA damage. Therapeutic intravitreal administration of 19,20-DHDP not only suppressed astrocyte loss but also reduced pathological vascular tuft formation in sEH–/– mice. Our data indicate that sEH activity is required for mitochondrial integrity and retinal astrocyte survival in ROP. Moreover, 19,20-DHDP may be more effective than DHA as a nutritional supplement at preventing retinopathy in preterm infants.
Jiong Hu, Sofia Iris Bibli, Janina Wittig, Sven Zukunft, Jihong Lin, Hans-Peter Hammes, Rüdiger Popp, Ingrid Fleming
There has been great progress in ocular gene therapy, but delivery of viral vectors to the retinal pigmented epithelium (RPE) and retina can be challenging. Subretinal injection, the preferred route of delivery for most applications, requires a surgical procedure that has risks. Herein we report a novel gene therapy delivery approach, suprachoroidal injection of AAV8 vectors, which is less invasive and could be done in an outpatient setting. Two weeks after suprachoroidal injection of AAV8.GFP in rats, GFP fluorescence covered 18.9% of RPE flat mounts and extended entirely around sagittal and transverse sections in RPE and photoreceptors. After 2 suprachoroidal injections of AAV8.GFP, GFP fluorescence covered 30.5% of RPE flat mounts. Similarly, widespread expression of GFP occurred in nonhuman primate and pig eyes after suprachoroidal injection of AAV8.GFP. Compared with subretinal injection in rats of RGX-314, an AAV8 vector expressing an anti-VEGF Fab, suprachoroidal injection of the same dose of RGX-314 resulted in similar expression of anti-VEGF Fab and similar suppression of VEGF-induced vascular leakage. Suprachoroidal AAV8 vector injection provides a noninvasive outpatient procedure to obtain widespread transgene expression in retina and RPE.
Kun Ding, Jikui Shen, Zibran Hafiz, Sean F. Hackett, Raquel Lima e Silva, Mahmood Khan, Valeria E. Lorenc, Daiqin Chen, Rishi Chadha, Minie Zhang, Sherri Van Everen, Nicholas Buss, Michele Fiscella, Olivier Danos, Peter A. Campochiaro
Inherited retinal degenerations are a common cause of untreatable blindness worldwide, with retinitis pigmentosa and cone dystrophy affecting approximately 1 in 3500 and 1 in 10,000 individuals, respectively. A major limitation to the development of effective therapies is the lack of availability of animal models that fully replicate the human condition. Particularly for cone disorders, rodent, canine, and feline models with no true macula have substantive limitations. By contrast, the cone-rich macula of a nonhuman primate (NHP) closely mirrors that of the human retina. Consequently, well-defined NHP models of heritable retinal diseases, particularly cone disorders that are predictive of human conditions, are necessary to more efficiently advance new therapies for patients. We have identified 4 related NHPs at the California National Primate Research Center with visual impairment and findings from clinical ophthalmic examination, advanced retinal imaging, and electrophysiology consistent with achromatopsia. Genetic sequencing confirmed a homozygous R565Q missense mutation in the catalytic domain of PDE6C, a cone-specific phototransduction enzyme associated with achromatopsia in humans. Biochemical studies demonstrate that the mutant mRNA is translated into a stable protein that displays normal cellular localization but is unable to hydrolyze cyclic GMP (cGMP). This NHP model of a cone disorder will not only serve as a therapeutic testing ground for achromatopsia gene replacement, but also for optimization of gene editing in the macula and of cone cell replacement in general.
Ala Moshiri, Rui Chen, Soohyun Kim, R. Alan Harris, Yumei Li, Muthuswamy Raveendran, Sarah Davis, Qingnan Liang, Ori Pomerantz, Jun Wang, Laura Garzel, Ashley Cameron, Glenn Yiu, J. Timothy Stout, Yijun Huang, Christopher J. Murphy, Jeffrey Roberts, Kota N. Gopalakrishna, Kimberly Boyd, Nikolai O. Artemyev, Jeffrey Rogers, Sara M. Thomasy
Mutations in CNGA3 and CNGB3, the genes encoding the subunits of the tetrameric cone photoreceptor cyclic nucleotide–gated ion channel, cause achromatopsia, a congenital retinal disorder characterized by loss of cone function. However, a small number of patients carrying the CNGB3/c.1208G>A;p.R403Q mutation present with a variable retinal phenotype ranging from complete and incomplete achromatopsia to moderate cone dysfunction or progressive cone dystrophy. By exploring a large patient cohort and published cases, we identified 16 unrelated individuals who were homozygous or (compound-)heterozygous for the CNGB3/c.1208G>A;p.R403Q mutation. In-depth genetic and clinical analysis revealed a co-occurrence of a mutant CNGA3 allele in a high proportion of these patients (10 of 16), likely contributing to the disease phenotype. To verify these findings, we generated a Cngb3R403Q/R403Q mouse model, which was crossbred with Cnga3-deficient (Cnga3–/–) mice to obtain triallelic Cnga3+/– Cngb3R403Q/R403Q mutants. As in human subjects, there was a striking genotype-phenotype correlation, since the presence of 1 Cnga3-null allele exacerbated the cone dystrophy phenotype in Cngb3R403Q/R403Q mice. These findings strongly suggest a digenic and triallelic inheritance pattern in a subset of patients with achromatopsia/severe cone dystrophy linked to the CNGB3/p.R403Q mutation, with important implications for diagnosis, prognosis, and genetic counseling.
Markus Burkard, Susanne Kohl, Timm Krätzig, Naoyuki Tanimoto, Christina Brennenstuhl, Anne E. Bausch, Katrin Junger, Peggy Reuter, Vithiyanjali Sothilingam, Susanne C. Beck, Gesine Huber, Xi-Qin Ding, Anja K. Mayer, Britta Baumann, Nicole Weisschuh, Ditta Zobor, Gesa-Astrid Hahn, Ulrich Kellner, Sascha Venturelli, Elvir Becirovic, Peter Charbel Issa, Robert K. Koenekoop, Günther Rudolph, John Heckenlively, Paul Sieving, Richard G. Weleber, Christian Hamel, Xiangang Zong, Martin Biel, Robert Lukowski, Matthias W. Seeliger, Stylianos Michalakis, Bernd Wissinger, Peter Ruth
Ciliopathies are clinically overlapping genetic disorders involving structural and functional abnormalities of cilia. Currently, there are no small-molecule drugs available to treat ciliary defects in ciliopathies. Our phenotype-based screen identified the flavonoid eupatilin and its analogs as lead compounds for developing ciliopathy medication. CEP290, a gene mutated in several ciliopathies, encodes a protein that forms a complex with NPHP5 to support the function of the ciliary transition zone. Eupatilin relieved ciliogenesis and ciliary receptor delivery defects resulting from deletion of CEP290. In rd16 mice harboring a blinding Cep290 in-frame deletion, eupatilin treatment improved both opsin transport to the photoreceptor outer segment and electrophysiological responses of the retina to light stimulation. The rescue effect was due to eupatilin-mediated inhibition of calmodulin binding to NPHP5, which promoted NPHP5 recruitment to the ciliary base. Our results suggest that deficiency of a ciliopathy protein could be mitigated by small-molecule compounds that target other ciliary components that interact with the ciliopathy protein.
Yong Joon Kim, Sungsoo Kim, Yooju Jung, Eunji Jung, Ho Jeong Kwon, Joon Kim