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BEST1: the Best Target for Gene and Cell Therapies
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Accepted Article Preview: Published ahead of advance online publication BEST1: the best target for gene and cell therapies
Tingting Yang, Sally Justus, Yao Li, Stephen H. Tsang
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Cite this article as: Tingting Yang, Sally Justus, Yao Li, Stephen H. Tsang, BEST1: the best target for gene and cell therapies, Molecular Therapy accepted article preview online 21 September 2015; doi:10.1038/mt.2015.177
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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
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Received 12 August 2015; accepted 14 September 2015; Accepted article preview online 21 September 2015
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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BEST1: the best target for gene and cell therapies Tingting Yang1, Sally Justus2, Yao Li2, 3, Stephen H. Tsang2, 3 1 2
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Department of Pharmacology & Physiology, University of Rochester, New York, New York, USA Barbara & Donald Jonas Stem Cell & Regenerative Medicine Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, New York, NY 10032
Irving Institute for Clinical and Translational Research, Columbia University Medical Center, New York, New York, USA
Correspondence should be addressed to SH Tsang: Stephen H. Tsang 635 West 165th Street, Box 112, New York, NY 10032 Phone: 001-212-342-1189 Fax: 001-212-342-4987 [email protected]
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BEST1: the best target for gene & cell therapies
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© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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Abstract
A retinal pigmented epithelial (RPE) disorder, bestrophinopathy has recently been proven to be amenable to gene and cell-based therapies in preclinical models. RPE disorders and allied retinal degenerations exhibit significant genetic heterogeneity, and diverse mutations can result in similar disease phenotypes. Several RPE disorders have recently become targets for gene therapies in humans. The year 2011 brought a new advance in cell-based therapies, with the FDA approving clinical trials using embryonic stem cells for an RPE disorder known as age-related macular
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degeneration. Recent studies on induced pluripotent stem (iPS)-RPE generation indicate strong
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potential for developing patient-specific disease models in vitro, which could eventually enable personalized treatment. This mini-review will briefly highlight the suitability of the retina for gene
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and cell therapies, the pathophysiology of bestrophinopathy, and the research and treatment opportunities afforded by stem cell and genetic therapies.
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Keywords: retinal-pigmented epithelial disorders, bestrophinopathy, BEST1, VMD, vitelliform, ARB, POS, CaCC, lipofuscin, iPSC
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The eye: an ideal environment for experimental gene and cell-based therapies
RPE disorders, including bestrophinopathy, some forms of retinitis pigmentosa, and age-related macular degeneration, affect more than 10 million Americans, an incidence that is expected to double by 2020 and will place a significant burden on public health.1 Aside from cancer, blindness is one of the most feared medical conditions among Americans,2 and for many patients, the diagnosis of an eye disease implies reduced independence and limited ability to conduct normal, day-to-day activities.
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Several of these debilitating blinding disorders have recently become targets for gene 3-7 and cell8
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therapies. There are currently twenty-six active and completed gene therapy trials (including RPE65 [MIM#613794], REP1 [MIM#300390], RS1 [MIM#300839], ABCA4 [MIM#248200], MYO7A
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[MIM#276900], MERTK [MIM#613862], RPGR [MIM#312610], CNGB3 [MIM#262300], and sFLT1 [MIM#165070]) and two embryonic stem cell (ESC) trials8, 9 for retinal degenerations.
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The human retina is the ideal testing ground for assessing experimental gene and cell therapies because of its relative immune privilege and accessibility for monitoring, imaging, and surgical manipulation.
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Numerous non-invasive techniques such as fundus autofluorescence, optical coherence tomography (OCT), and adaptive optics imaging enable micron-level examination of the inside of the retina in live animals and patients. The ability to quantify efficacy in subjects in a non-invasive manner at multiple time points is a major advantage of conducting research on the eye, one shared by few if any other organ systems. In fact, the eye is one of the only organs that permits remarkably targeted interventions that are not highly invasive or systemic. The blood-retina barrier provides relative immune privilege, and in the event of teratoma formation, the eye can be removed without risk of damaging other organ systems and is itself not a life-threatening event. Experimentally, eyes provide the ideal treatment-control groups, as the contralateral, untreated eye can be compared with the intervention eye.
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Quantifiable therapeutic endpoints and the long therapeutic window for bestrophinopathies
Bestrophinopathies are caused by mutations in the gene encoding BESTROPHIN1 (BEST1 [MIM#607854]), with over 120 amino acid substitution mutations associated with the disease.10,
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Diagnosis is typically made based on fundus appearance, EOG measurements, and family history.12, 13 A decreased EOG Arden ratio (light peak divided by dark trough) is a hallmark of all bestrophinopathies and is caused by malfunctions in the protein bestrophin-1, believed to be a Ca2+-regulated chloride
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channel (CaCC) in the basolateral membrane of the RPE.14 Figures 1 through 4 provide fundus and/or
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OCT examples ranging from healthy individuals (Fig. 1) to those with severe bestrophinopathy (Fig. 4).
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While bestrophinopathies progressively worsen with age, the rate of decline is typically slow and provides a long therapeutic window, as central photoreceptors remain viable for decades despite the
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persistence of subretinal fluid and serous detachment (Figs. 2 and 3). Most bestrophinopathy patients
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will maintain robust central vision well into the fifth decade of life15, as cones can tolerate subretinal fluid for many years.16 Eventually rod cell death (Fig. 4) due to subretinal fluid will cause cones to
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degenerate soon after16-18, which results in blindness and cannot be treated with gene supplementation therapy. Thus, unless rod transplantation is applied, the ideal time for intervention is as soon as possible after diagnosis or at least before fluid-induced rod cell death.
Quantifiable therapeutic endpoints are readily obtainable for bestrophinopathies, which feature subretinal fluid accumulation. Fluid resolution on OCT thickness measurements (in microns) provides sensitive therapeutic endpoints and can reduce the required sample size in a gene therapy trial. Contrastingly, cell preservation is the standard metric in many ongoing human gene therapy trials for retinal degeneration, even though this method is a much more challenging outcome measurement to
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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achieve than quantifying fluid resolution. The relative ease of appraising disease development or treatment progress is more closely comparable to that of exudative age-related macular degeneration trials using OCT anatomical outcome measurements, where subretinal fluid can be resolved within a week after treatment.
An autosomal dominant bestrophinopathy known as Best Vitelliform Macular Degeneration (VMD [MIM#153700]) is a debilitating, early-onset form of central macular degeneration. While rod and cone photoreceptor function remains unaffected, the disease leads to the formation of serous retinal
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detachment and lesions that resemble egg yolk, or vitelliform, due to abnormalities in the fluid and/or
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electrolyte homeostasis between the RPE and photoreceptor outer segments (Figs. 2-4).
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Normally, the RPE phagocytoses photoreceptor outer segments (POS), which are composed of lipid and 14
oxidized protein wastes, to allow cell renewal. Phagocytosis is affected by ion and fluid flux, which is
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believed to be under the control of bestrophin-1.19 Although the mechanism is still unclear, mutations in
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BEST1 alter the CaCC conductance of the bestrophin-1 protein, which hinders the RPE from digesting the POS waste products.10 The accumulation of this bisretinoid waste is termed lipofuscin, which has
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toxic effects on the RPE and is directly correlated with the severity of macular degeneration.10, 15 Consequently, the abnormal appearance of the macula in bestrophinopathy patients is caused by the autofluorescent properties of lipofuscin (Fig. 2b). The ability to model this mechanism in the dish could provide unique opportunities for testing drug-like compounds that improve the rate of phagocytosis in iPS-derived, diseased RPE.
Autosomal recessive bestrophinopathy (ARB) is a similar disorder to VMD but differs in its inheritance pattern and some phenotypic manifestations, and it results in a nearly nonfunctional bestrophin-1 protein.20 ARB is caused by loss-of-function alleles21, 22 and is characterized by multifocal vitelliform
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dystrophy with subretinal fluid (Fig. 5). One of patients’ first symptoms is central vision loss with variable onset, and they are typically hyperopic, exhibiting bilateral multifocal hyper-autofluorescent vitelliform lesions and shallow serous retinal detachment.20 Scotopic and photopic full-field ERGs usually diminish with age and the EOG shows a reduced or absent light rise. Thus, one distinguishing diagnostic feature between VMD and ARB is that individuals with VMD typically have normal fullfield ERGs while ARB patients do not.20, 23
Crystallography allows targeted drug design and precision medicine
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Despite bestrophin-1’s clear pathological relevance to bestrophinopathy diseases24, the physiological role and disease-causing mechanisms of bestrophin-1 remain elusive. Therefore, dissecting the structural
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and functional properties of this protein would be a significant step towards understanding bestrophinopathies and developing treatment options. Recently, the crystal structures of a bacterial
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(Klebsiella pneumoniae, KpBest) and a chicken bestrophin-1 homolog were solved independently.25, 26
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The structure models reveal that bestrophin-1 forms a stable pentamer, with each protomer containing four transmembrane helices and cytosolic N- and C-termini. The ion permeation pathway has a flower-
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vase shape with two permeation restrictions. Electrophysiological analysis of structure-inspired mutations demonstrated that the first restriction is a sensitive control of ion selectivity, while the second restriction is an activation gate in both KpBest and human bestrophin-1, showcasing the power of crystallography in inspiring functional studies.
Importantly, the crystallography of bestrophin-1 potentiates targeted drug design for bestrophinopathies by predicting the disease-causing mechanism of a given BEST1 mutation in a patient and providing a structural platform for small molecule-based drug screens. To understand characteristics of human bestrophin-1 and the disposition of disease-causing mutations, a homology model for its transmembrane
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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portion was generated based on the KpBest structure. When plotting disease-causing mutations in human BEST1 onto the three-dimensional (3D) model, the distribution of mutations suggests that there may be multiple disease-causing mechanisms.25 For example, mutations along the ion-conducting pathway (e.g. F80 and I205) may affect Cl- conduction, while mutations around a carboxylate loop, which is a possible Ca2+ binding domain (e.g. E300 and D302), may disrupt channel activation, as the channel is Ca2+-activated. The 3D structure of wild type bestrophin-1 or patient-derived mutants will guide drug discovery and personalized medicine in the near future. Compounds can be screened and designed on the computer based on the predicted interacting interface of a desired structural/functional
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domain of bestrophin-1. Then the binding mode of synthesized compounds to bestrophin-1 will be
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examined by X-ray crystallography for confirmation, followed by further validation and optimization. In short, developing precision medicine-based approaches for bestrophinopathy patients is contingent on
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understanding the structure and function of bestrophin-1.
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Treatment strategies: AAV, animal models, ESC, iPSC
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VMD is likely due to a dominant-gene effect wherein the expression of the mutated protein interferes
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with the CaCC activity of wild-type bestrophin-121, 27 and/or the regulation of other ion channels.14 If VMD mutations simply antagonize wild-type bestrophin-1 function, AAV-mediated ribozyme and geneediting delivery will be needed for correction, and subretinal fluid can be resolved by restoration of the CaCC conductance of bestrophin-1 through gene or cell supplementation. On the other hand, recessive, loss-of-function alleles, as in ARB, can be corrected by gene supplementation, in which a single wild type copy is transduced into the affected cells. An alternative expression hypothesis, haploinsufficieny was not supported by data presented on the parents of five ARB patients from different families28. Of note, one of the five ARB patients28 had homozygous null alleles, yet her parents had no phenotypic manifestations, which further supports the notion of a dominant-negative effect of VMD.
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Until the development of patient-specific stem cell lines, progress to therapy was stalled because Best1 knockout mice and knockin mice do not recapitulate diseased human phenotypes.29 In contrast to expectation, mice homozygous for a Best1 knockout mutation do not exhibit abnormal EOG responses or an eye phenotype that resembles human VMD.30 Furthermore, these knockout mice show normal RPE CaCC activity, indicating that mouse bestrophin-1 is not a major contributor to CaCC function in mouse RPE. Overexpression and knockdown of Best1 in rat RPE also does not have the expected effects. Contrastingly, a dog model of ARB called canine multifocal retinopathy (cmr) accurately
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replicated human ARB and was also treatable through genetic intervention: fundus lesions were resolved
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for at least 23 months post-rAAV2/2 injection, although improvement in ERG has yet to be reported. Thus, while dominant forms of the disorder may be more difficult to model in animals, canine models of
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the recessive forms are already successfully being treated by overriding the mutations responsible for them.
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An alternative to animal models, stem cells can readily be obtained from patient fibroblasts and differentiated into RPE, which may provide a useful experimental system to study bestrophin-1 function.
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In a recent study, patient-specific RPE derived from two VMD patients showed delayed rhodopsin degradation and decreased Ca2+-stimulated responses in phagocytosis assays, as compared to unaffected sibling controls.19 With the employment of VMD patient-derived RPE, techniques to correct the genetic defect may be developed to treat the disease on a patient-specific basis.32, 33 While there are also clinical trials currently underway to determine whether ESCs can replace diseased RPE, the advantage of iPS therapies is that genetically-repaired iPS cells allow for autologous transplantation, which does not require immunosuppression therapy. Successful restoration of CaCC conductance in patient-specific iPS-RPE can be an indicator of the efficacy of this therapeutic approach in subsequent clinical trials.
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Gene Repair and Precision Medicine
Until recently, retinal degenerations including bestrophinopathy faced overwhelming hurdles to treatment, as their pathophysiology and crystallographic structure were unavailable. Now, it is feasible to identity each patient’s genotype and corresponding mutation, create autologous iPS-RPE, replace the mutated allele with the wild type form, and implant the healthy cells into the retina. Significant advances in our understanding of BEST1 and its involvement in bestrophinopathy must be made in order to refine and optimize this treatment approach, but the future of personalized medicine is quickly approaching
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and soon to be within reach.
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Acknowledgements
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The authors declare no conflict of interest. The Barbara & Donald Jonas Stem Cell & Regenerative
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Medicine Laboratory is supported by NIH R01EY018213, R01EY024698, and R21AG050437; the
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Research to Prevent Blindness Physician-Scientist Award; New York State (C029572); and the Foundation Fighting Blindness New York Regional Research Center Grant (C-NY05-0705-0312).
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Additionally, the authors would like to extend their gratitude and appreciation to our patients and especially to Lawrence Chan for reviewing this manuscript as well as serving as our leading research adviser for our macular degeneration program.
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Figure Captions: Figure 1. Healthy Eyes. The fundus autofluoresence (FA) (a) and spectral-domain optical coherence tomography (SD-OCT) (b) of a healthy individual are provided for comparison with disease states. The white arrows indicate the photoreceptor mitochondrial band.
Figure 2. Early bestrophinopathy vitelliform macular degeneration (VMD [MIM#153700]). Mild phenotypic manifestation in a 5 year old VMD patient with a BEST1, p. Ala10Thr mutation. Color fundus photograph of the left eye shows the vitelliform lesion in the central macula (a). Subretinal fluid*
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and outer segment debris are visible in the SD-OCT images (b). The central opaque region corresponds
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to an area of serous detachment. Photoreceptor outer segments (POS) are hyper-autofluorescent in FA imaging. *= subretinal fluid; black arrows = photoreceptor mitochondrial band
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Figure 3. Moderate VMD. Moderate phenotypic manifestation in a 58 year old VMD patient with a
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novel p. Asp302Ala mutation. The vitelliform is pronounced in the color fundus imaging (a), the
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photoreceptor layer is thin compared to that of a healthy individual, and extensive fluid has developed subretinally in OCT imaging (b). *= subretinal fluid; black arrows = photoreceptor mitochondrial band.
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Figure 4. Severe VMD. Severe phenotypic manifestation in a 42 year old VMD patient with p. Asp302Ala (novel) and p. Arg218His mutations. Photoreceptor mitochondrial band has disintegrated at various points, indicated by black arrows. FA is absent in the central area, which exhibits dystrophic RPE and loss of photoreceptors.
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Figure 5. Autosomal recessive bestrophinopathy (ARB [MIM#611809 ]). (a): Right eye; (b): Left eye. Color montage fundus photographs are from a 6 year old ARB patient with the homozygous mutation p. Phe283del. In the right eye, there is vitelliform material nasal to the optic disc, while the left eye exhibits multifocal, curvilinear, yellowish subretinal deposits along the superotemporal arcade.
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Accepted Article Preview: Published ahead of advance online publication BEST1: the best target for gene and cell therapies
Tingting Yang, Sally Justus, Yao Li, Stephen H. Tsang
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Cite this article as: Tingting Yang, Sally Justus, Yao Li, Stephen H. Tsang, BEST1: the best target for gene and cell therapies, Molecular Therapy accepted article preview online 21 September 2015; doi:10.1038/mt.2015.177
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This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG is providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
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Received 12 August 2015; accepted 14 September 2015; Accepted article preview online 21 September 2015
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BEST1: the best target for gene and cell therapies Tingting Yang1, Sally Justus2, Yao Li2, 3, Stephen H. Tsang2, 3 1 2
3
Department of Pharmacology & Physiology, University of Rochester, New York, New York, USA Barbara & Donald Jonas Stem Cell & Regenerative Medicine Laboratory, and Bernard & Shirlee Brown Glaucoma Laboratory, Department of Pathology & Cell Biology, Institute of Human Nutrition, College of Physicians and Surgeons, Columbia University, New York, NY 10032
Irving Institute for Clinical and Translational Research, Columbia University Medical Center, New York, New York, USA
Correspondence should be addressed to SH Tsang: Stephen H. Tsang 635 West 165th Street, Box 112, New York, NY 10032 Phone: 001-212-342-1189 Fax: 001-212-342-4987 [email protected]
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BEST1: the best target for gene & cell therapies
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© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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Abstract
A retinal pigmented epithelial (RPE) disorder, bestrophinopathy has recently been proven to be amenable to gene and cell-based therapies in preclinical models. RPE disorders and allied retinal degenerations exhibit significant genetic heterogeneity, and diverse mutations can result in similar disease phenotypes. Several RPE disorders have recently become targets for gene therapies in humans. The year 2011 brought a new advance in cell-based therapies, with the FDA approving clinical trials using embryonic stem cells for an RPE disorder known as age-related macular
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degeneration. Recent studies on induced pluripotent stem (iPS)-RPE generation indicate strong
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potential for developing patient-specific disease models in vitro, which could eventually enable personalized treatment. This mini-review will briefly highlight the suitability of the retina for gene
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and cell therapies, the pathophysiology of bestrophinopathy, and the research and treatment opportunities afforded by stem cell and genetic therapies.
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Keywords: retinal-pigmented epithelial disorders, bestrophinopathy, BEST1, VMD, vitelliform, ARB, POS, CaCC, lipofuscin, iPSC
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© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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The eye: an ideal environment for experimental gene and cell-based therapies
RPE disorders, including bestrophinopathy, some forms of retinitis pigmentosa, and age-related macular degeneration, affect more than 10 million Americans, an incidence that is expected to double by 2020 and will place a significant burden on public health.1 Aside from cancer, blindness is one of the most feared medical conditions among Americans,2 and for many patients, the diagnosis of an eye disease implies reduced independence and limited ability to conduct normal, day-to-day activities.
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Several of these debilitating blinding disorders have recently become targets for gene 3-7 and cell8
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therapies. There are currently twenty-six active and completed gene therapy trials (including RPE65 [MIM#613794], REP1 [MIM#300390], RS1 [MIM#300839], ABCA4 [MIM#248200], MYO7A
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[MIM#276900], MERTK [MIM#613862], RPGR [MIM#312610], CNGB3 [MIM#262300], and sFLT1 [MIM#165070]) and two embryonic stem cell (ESC) trials8, 9 for retinal degenerations.
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The human retina is the ideal testing ground for assessing experimental gene and cell therapies because of its relative immune privilege and accessibility for monitoring, imaging, and surgical manipulation.
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Numerous non-invasive techniques such as fundus autofluorescence, optical coherence tomography (OCT), and adaptive optics imaging enable micron-level examination of the inside of the retina in live animals and patients. The ability to quantify efficacy in subjects in a non-invasive manner at multiple time points is a major advantage of conducting research on the eye, one shared by few if any other organ systems. In fact, the eye is one of the only organs that permits remarkably targeted interventions that are not highly invasive or systemic. The blood-retina barrier provides relative immune privilege, and in the event of teratoma formation, the eye can be removed without risk of damaging other organ systems and is itself not a life-threatening event. Experimentally, eyes provide the ideal treatment-control groups, as the contralateral, untreated eye can be compared with the intervention eye.
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Quantifiable therapeutic endpoints and the long therapeutic window for bestrophinopathies
Bestrophinopathies are caused by mutations in the gene encoding BESTROPHIN1 (BEST1 [MIM#607854]), with over 120 amino acid substitution mutations associated with the disease.10,
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Diagnosis is typically made based on fundus appearance, EOG measurements, and family history.12, 13 A decreased EOG Arden ratio (light peak divided by dark trough) is a hallmark of all bestrophinopathies and is caused by malfunctions in the protein bestrophin-1, believed to be a Ca2+-regulated chloride
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channel (CaCC) in the basolateral membrane of the RPE.14 Figures 1 through 4 provide fundus and/or
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OCT examples ranging from healthy individuals (Fig. 1) to those with severe bestrophinopathy (Fig. 4).
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While bestrophinopathies progressively worsen with age, the rate of decline is typically slow and provides a long therapeutic window, as central photoreceptors remain viable for decades despite the
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persistence of subretinal fluid and serous detachment (Figs. 2 and 3). Most bestrophinopathy patients
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will maintain robust central vision well into the fifth decade of life15, as cones can tolerate subretinal fluid for many years.16 Eventually rod cell death (Fig. 4) due to subretinal fluid will cause cones to
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degenerate soon after16-18, which results in blindness and cannot be treated with gene supplementation therapy. Thus, unless rod transplantation is applied, the ideal time for intervention is as soon as possible after diagnosis or at least before fluid-induced rod cell death.
Quantifiable therapeutic endpoints are readily obtainable for bestrophinopathies, which feature subretinal fluid accumulation. Fluid resolution on OCT thickness measurements (in microns) provides sensitive therapeutic endpoints and can reduce the required sample size in a gene therapy trial. Contrastingly, cell preservation is the standard metric in many ongoing human gene therapy trials for retinal degeneration, even though this method is a much more challenging outcome measurement to
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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achieve than quantifying fluid resolution. The relative ease of appraising disease development or treatment progress is more closely comparable to that of exudative age-related macular degeneration trials using OCT anatomical outcome measurements, where subretinal fluid can be resolved within a week after treatment.
An autosomal dominant bestrophinopathy known as Best Vitelliform Macular Degeneration (VMD [MIM#153700]) is a debilitating, early-onset form of central macular degeneration. While rod and cone photoreceptor function remains unaffected, the disease leads to the formation of serous retinal
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detachment and lesions that resemble egg yolk, or vitelliform, due to abnormalities in the fluid and/or
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electrolyte homeostasis between the RPE and photoreceptor outer segments (Figs. 2-4).
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Normally, the RPE phagocytoses photoreceptor outer segments (POS), which are composed of lipid and 14
oxidized protein wastes, to allow cell renewal. Phagocytosis is affected by ion and fluid flux, which is
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believed to be under the control of bestrophin-1.19 Although the mechanism is still unclear, mutations in
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BEST1 alter the CaCC conductance of the bestrophin-1 protein, which hinders the RPE from digesting the POS waste products.10 The accumulation of this bisretinoid waste is termed lipofuscin, which has
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toxic effects on the RPE and is directly correlated with the severity of macular degeneration.10, 15 Consequently, the abnormal appearance of the macula in bestrophinopathy patients is caused by the autofluorescent properties of lipofuscin (Fig. 2b). The ability to model this mechanism in the dish could provide unique opportunities for testing drug-like compounds that improve the rate of phagocytosis in iPS-derived, diseased RPE.
Autosomal recessive bestrophinopathy (ARB) is a similar disorder to VMD but differs in its inheritance pattern and some phenotypic manifestations, and it results in a nearly nonfunctional bestrophin-1 protein.20 ARB is caused by loss-of-function alleles21, 22 and is characterized by multifocal vitelliform
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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dystrophy with subretinal fluid (Fig. 5). One of patients’ first symptoms is central vision loss with variable onset, and they are typically hyperopic, exhibiting bilateral multifocal hyper-autofluorescent vitelliform lesions and shallow serous retinal detachment.20 Scotopic and photopic full-field ERGs usually diminish with age and the EOG shows a reduced or absent light rise. Thus, one distinguishing diagnostic feature between VMD and ARB is that individuals with VMD typically have normal fullfield ERGs while ARB patients do not.20, 23
Crystallography allows targeted drug design and precision medicine
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Despite bestrophin-1’s clear pathological relevance to bestrophinopathy diseases24, the physiological role and disease-causing mechanisms of bestrophin-1 remain elusive. Therefore, dissecting the structural
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and functional properties of this protein would be a significant step towards understanding bestrophinopathies and developing treatment options. Recently, the crystal structures of a bacterial
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(Klebsiella pneumoniae, KpBest) and a chicken bestrophin-1 homolog were solved independently.25, 26
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The structure models reveal that bestrophin-1 forms a stable pentamer, with each protomer containing four transmembrane helices and cytosolic N- and C-termini. The ion permeation pathway has a flower-
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vase shape with two permeation restrictions. Electrophysiological analysis of structure-inspired mutations demonstrated that the first restriction is a sensitive control of ion selectivity, while the second restriction is an activation gate in both KpBest and human bestrophin-1, showcasing the power of crystallography in inspiring functional studies.
Importantly, the crystallography of bestrophin-1 potentiates targeted drug design for bestrophinopathies by predicting the disease-causing mechanism of a given BEST1 mutation in a patient and providing a structural platform for small molecule-based drug screens. To understand characteristics of human bestrophin-1 and the disposition of disease-causing mutations, a homology model for its transmembrane
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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portion was generated based on the KpBest structure. When plotting disease-causing mutations in human BEST1 onto the three-dimensional (3D) model, the distribution of mutations suggests that there may be multiple disease-causing mechanisms.25 For example, mutations along the ion-conducting pathway (e.g. F80 and I205) may affect Cl- conduction, while mutations around a carboxylate loop, which is a possible Ca2+ binding domain (e.g. E300 and D302), may disrupt channel activation, as the channel is Ca2+-activated. The 3D structure of wild type bestrophin-1 or patient-derived mutants will guide drug discovery and personalized medicine in the near future. Compounds can be screened and designed on the computer based on the predicted interacting interface of a desired structural/functional
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domain of bestrophin-1. Then the binding mode of synthesized compounds to bestrophin-1 will be
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examined by X-ray crystallography for confirmation, followed by further validation and optimization. In short, developing precision medicine-based approaches for bestrophinopathy patients is contingent on
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understanding the structure and function of bestrophin-1.
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Treatment strategies: AAV, animal models, ESC, iPSC
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VMD is likely due to a dominant-gene effect wherein the expression of the mutated protein interferes
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with the CaCC activity of wild-type bestrophin-121, 27 and/or the regulation of other ion channels.14 If VMD mutations simply antagonize wild-type bestrophin-1 function, AAV-mediated ribozyme and geneediting delivery will be needed for correction, and subretinal fluid can be resolved by restoration of the CaCC conductance of bestrophin-1 through gene or cell supplementation. On the other hand, recessive, loss-of-function alleles, as in ARB, can be corrected by gene supplementation, in which a single wild type copy is transduced into the affected cells. An alternative expression hypothesis, haploinsufficieny was not supported by data presented on the parents of five ARB patients from different families28. Of note, one of the five ARB patients28 had homozygous null alleles, yet her parents had no phenotypic manifestations, which further supports the notion of a dominant-negative effect of VMD.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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Until the development of patient-specific stem cell lines, progress to therapy was stalled because Best1 knockout mice and knockin mice do not recapitulate diseased human phenotypes.29 In contrast to expectation, mice homozygous for a Best1 knockout mutation do not exhibit abnormal EOG responses or an eye phenotype that resembles human VMD.30 Furthermore, these knockout mice show normal RPE CaCC activity, indicating that mouse bestrophin-1 is not a major contributor to CaCC function in mouse RPE. Overexpression and knockdown of Best1 in rat RPE also does not have the expected effects. Contrastingly, a dog model of ARB called canine multifocal retinopathy (cmr) accurately
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replicated human ARB and was also treatable through genetic intervention: fundus lesions were resolved
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for at least 23 months post-rAAV2/2 injection, although improvement in ERG has yet to be reported. Thus, while dominant forms of the disorder may be more difficult to model in animals, canine models of
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the recessive forms are already successfully being treated by overriding the mutations responsible for them.
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An alternative to animal models, stem cells can readily be obtained from patient fibroblasts and differentiated into RPE, which may provide a useful experimental system to study bestrophin-1 function.
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In a recent study, patient-specific RPE derived from two VMD patients showed delayed rhodopsin degradation and decreased Ca2+-stimulated responses in phagocytosis assays, as compared to unaffected sibling controls.19 With the employment of VMD patient-derived RPE, techniques to correct the genetic defect may be developed to treat the disease on a patient-specific basis.32, 33 While there are also clinical trials currently underway to determine whether ESCs can replace diseased RPE, the advantage of iPS therapies is that genetically-repaired iPS cells allow for autologous transplantation, which does not require immunosuppression therapy. Successful restoration of CaCC conductance in patient-specific iPS-RPE can be an indicator of the efficacy of this therapeutic approach in subsequent clinical trials.
© 2015 The American Society of Gene & Cell Therapy. All rights reserved
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Gene Repair and Precision Medicine
Until recently, retinal degenerations including bestrophinopathy faced overwhelming hurdles to treatment, as their pathophysiology and crystallographic structure were unavailable. Now, it is feasible to identity each patient’s genotype and corresponding mutation, create autologous iPS-RPE, replace the mutated allele with the wild type form, and implant the healthy cells into the retina. Significant advances in our understanding of BEST1 and its involvement in bestrophinopathy must be made in order to refine and optimize this treatment approach, but the future of personalized medicine is quickly approaching
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and soon to be within reach.
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Acknowledgements
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The authors declare no conflict of interest. The Barbara & Donald Jonas Stem Cell & Regenerative
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Medicine Laboratory is supported by NIH R01EY018213, R01EY024698, and R21AG050437; the
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Research to Prevent Blindness Physician-Scientist Award; New York State (C029572); and the Foundation Fighting Blindness New York Regional Research Center Grant (C-NY05-0705-0312).
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Additionally, the authors would like to extend their gratitude and appreciation to our patients and especially to Lawrence Chan for reviewing this manuscript as well as serving as our leading research adviser for our macular degeneration program.
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Figure Captions: Figure 1. Healthy Eyes. The fundus autofluoresence (FA) (a) and spectral-domain optical coherence tomography (SD-OCT) (b) of a healthy individual are provided for comparison with disease states. The white arrows indicate the photoreceptor mitochondrial band.
Figure 2. Early bestrophinopathy vitelliform macular degeneration (VMD [MIM#153700]). Mild phenotypic manifestation in a 5 year old VMD patient with a BEST1, p. Ala10Thr mutation. Color fundus photograph of the left eye shows the vitelliform lesion in the central macula (a). Subretinal fluid*
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and outer segment debris are visible in the SD-OCT images (b). The central opaque region corresponds
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to an area of serous detachment. Photoreceptor outer segments (POS) are hyper-autofluorescent in FA imaging. *= subretinal fluid; black arrows = photoreceptor mitochondrial band
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Figure 3. Moderate VMD. Moderate phenotypic manifestation in a 58 year old VMD patient with a
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novel p. Asp302Ala mutation. The vitelliform is pronounced in the color fundus imaging (a), the
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photoreceptor layer is thin compared to that of a healthy individual, and extensive fluid has developed subretinally in OCT imaging (b). *= subretinal fluid; black arrows = photoreceptor mitochondrial band.
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Figure 4. Severe VMD. Severe phenotypic manifestation in a 42 year old VMD patient with p. Asp302Ala (novel) and p. Arg218His mutations. Photoreceptor mitochondrial band has disintegrated at various points, indicated by black arrows. FA is absent in the central area, which exhibits dystrophic RPE and loss of photoreceptors.
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Figure 5. Autosomal recessive bestrophinopathy (ARB [MIM#611809 ]). (a): Right eye; (b): Left eye. Color montage fundus photographs are from a 6 year old ARB patient with the homozygous mutation p. Phe283del. In the right eye, there is vitelliform material nasal to the optic disc, while the left eye exhibits multifocal, curvilinear, yellowish subretinal deposits along the superotemporal arcade.
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Figure 5
© 2015 The American Society of Gene & Cell Therapy. All rights reserved