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Progress in Retinal and Eye Research xxx (2014) 1e21 Contents lists available at ScienceDirect Progress in Retinal and Eye Research journal homepage: www.elsevier.com/locate/prer Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases Luke A. Wiley a, 1, 2, Erin R. Burnight a, 1, 2, Allison E. Songstad a, 1, 2, Arlene V. Drack a, 1, Robert F. Mullins a, 1, Edwin M. Stone a, b, 1, Budd A. Tucker a, *, 1 a b Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USA Howard Hughes Medical Institute, University of Iowa, Iowa City, IA, USA a r t i c l e i n f o a b s t r a c t Article history: Received 31 July 2014 Received in revised form 15 October 2014 Accepted 16 October 2014 Available online xxx Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness is one of people's most feared maladies. Heritable diseases of the retina, such as age-related macular degeneration and retinitis pigmentosa, are the leading cause of blindness in the developed world, collectively affecting as many as one-third of all people over the age of 75, to some degree. For decades, scientists have dreamed of preventing vision loss or of restoring the vision of patients affected with retinal degeneration through drug therapy, gene augmentation or a cell-based transplantation approach. In this review we will discuss the use of the induced pluripotent stem cell technology to model and develop various treatment modalities for the treatment of inherited retinal degenerative disease. We will focus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene therapeutics and as a source of autologous cells for cell transplantation and replacement. © 2014 Published by Elsevier Ltd. Keywords: Induced pluripotent stem cells Retinal degeneration Gene therapy CRISPR-based genome editing Cell transplantation Contents 1. 2. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1. Stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1. Cellular potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2. Origin of isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2. Induced pluripotent stem cells (iPSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Disease modeling using iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.1. Mendelian retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.2. MAK-associated retinitis pigmentosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3. Usher syndrome type II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 00 00 00 00 00 00 00 00 List of abbreviations: AMD, age-related macular degeneration; RPE, retinal pigmented epithelium; ESC, embryonic stem cell; ASC, adult stem cell; iPSC, induced pluripotent stem cell; RP, retinitis pigmentosa; AAV, adeno-associated virus; LCA, Leber congenital amaurosis; TALEN, transcription activator-like effector nuclease; CRISPR, clustered regulatory interspaced short palindromic repeat; ZFN, zinc finger nuclease; sgRNA, small guide RNA; DSB, double-strand break; NHEJ, non-homologous end joining; InDel, insertion or deletion; HDR, homology-directed repair; RPC, retinal progenitor cell; ACAID, anterior chamber-associated immune deviation. * Corresponding author. Stephen A. Wynn Institute for Vision Research, Carver College of Medicine, University of Iowa, Department of Ophthalmology and Visual Sciences, 375 Newton Road, Iowa City, IA 52242, USA. Tel.: þ1 319 355 7242. E-mail address: [email protected] (B.A. Tucker). 1 Percentage of work contributed by each author in the production of the manuscript is as follows: Luke A. Wiley: 20%; Erin R. Burnight: 20%; Allison E. Songstad: 20%; Arlene V. Drack: 7.5%; Robert F. Mullins: 7.5%; Edwin M. Stone: 7.5%; Budd A. Tucker: 17.5%. 2 These authors contributed equally to this manuscript. http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 1350-9462/© 2014 Published by Elsevier Ltd. Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 2 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 3. 4. 5. 2.4. CEP290-associated Leber congenital amaurosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.5. Juvenile neuronal ceroid lipofuscinosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.6. Best disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.7. Using iPSCs to model AMD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Gene augmentation and cell replacement treatment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. iPSCs and gene augmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. iPSCs and genome editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3. iPSCs for testing of therapeutic efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4. iPSCs and drug discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.5. Retinal transplantation and cellular replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.6. Retinal immune landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Introduction Heritable retinal degenerative diseases, which include Mendelian disorders such as retinitis pigmentosa, Stargardt disease and Leber congenital amaurosis, as well as the more complex and heterogeneous disease, age-related macular degeneration (AMD), are collectively the leading cause of incurable blindness in the developed world (Huang et al., 2011). For years, ophthalmologists and vision scientists have dreamed of restoring vision to those that have lost this precious sense. Fortunately, despite widespread death of the photoreceptors, retinal pigmented epithelium (RPE) and choroidal endothelial cells e which comprise the outer retina– the inner retina, which consists of ganglion cells, bipolar cells, amacrine cells, horizontal cells, and Müller glia is largely spared in these diseases (Huang et al., 2011; Mullins et al., 2009, 2012). Preservation of the inner neural retina presents an opportunistic environment in which preservation and replacement of these outer retinal cell populations could maintain and restore visual function. Over the past decade, studies employing stem cells for disease modeling and treatment of incurable diseases have gained momentum in the field of regenerative medicine. The groundwork of the stem cell field goes back more than 50 years when Sir John Gurdon demonstrated that nuclear transfer of tadpole nuclei into a recipient oocyte resulted in the generation of clonally identical frogs (Gurdon, 1962). In contradiction to the concept classically illustrated by Conrad Waddington's proposal of the ‘epigenetic landscape,’ re-depicted here in Fig. 1, this work demonstrated that the developed characteristics of somatic cells are not fixed (Ladewig et al., 2013; Waddington, 1957). That is, unlike Weismann's barrier theory, which stated that unwarranted genetic information is erased in cells committed to a certain state (Weismann, 1893), Gurdon's nuclear transfer experiments showed that not only do somatic cells maintain all genetic material, but they can also be reignited by artificial manipulations allowing for the return to a state of pluripotency. Shortly after Gurdon's groundbreaking work with nuclear transfer, many other groups started to present more novel findings pertaining to stem cells. Dr. James Till and Dr. Ernest McCulloch provided data to prove the existence of stem cells by injecting healthy marrow cells into irradiated mice and observing the presence of undifferentiated cellular colonies on the recipients' spleens (Till and McCulloch, 1961). Two decades after Till and McCulloch's finding, Drs. Martin Evans and Matthew Kaufman reported their ability to successfully culture pluripotent cell lines from mouse blastocysts and differentiate these cells both in vitro and in vivo (Evans and Kaufman, 1981). In the following decade, Dr. Ian Wilmut made international headlines 00 00 00 00 00 00 00 00 00 00 00 00 00 00 00 when he used Dr. Gurdon's nuclear transfer method to clone Dolly the sheep by replacing the nucleus of a fertilized embryo with the nucleus of an adult mammary gland cell (Campbell et al., 1996). In 1998 Dr. James Thomson further contributed to the stem cell field by successfully isolating human embryonic stem cells (Thomson et al., 1998). Together, these findings along with many other reports paved the way for modern stem cell research and regenerative medicine. 1.1. Stem cells Stem cells are defined as cells that have an unlimited capacity for self-renewal and are capable of differentiating into multiple cell types. They are often categorized according to 1) potency, or the Fig. 1. The epigenetic landscape of somatic cells. Conrad Waddington proposed that pluripotent stem cells terminally differentiate into somatic cells. It is now known that pluripotent stem cells undergo stepwise differentiation (blue arrows), producing lineage-specific progenitor cells that go on to further differentiate into germ layerspecific progenitor cells (EndoSP, MesoSP and EctoSP). These layer-specific progenitor cells then produce cells that comprise germ layer-specific organs of the endoderm (liver), mesoderm (heart) and ectoderm (brain), to name a few examples. Stem cell research pioneers, like Gurdon and Yamanaka, demonstrated that somatic cells from any germ layer could be reprogrammed into pluripotent stem cells (green arrow) and directly converted from one somatic cell type into another somatic cell type (purple arrow). PS: pluripotent stem cell, LSP: lineage-specific progenitor, EndoSP: endodermspecific progenitor, MesoSP: mesoderm-specific progenitor, EctoSP: ectoderm-specific progenitor. Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 3 degree or capacity in which they can differentiate into multiple cells types or lineages, and 2) origin of isolation. immune suppression may be required to prevent immunemediated rejection. 1.1.1. Cellular potency Totipotent stem cells have unlimited capacity and are thus able to differentiate into either embryonic or extra-embryonic tissues. Pluripotent stem cells have the capacity to form embryonic tissue and thus the embryonic cellular lineages: ectoderm, mesoderm, and endoderm. Multipotent stem cells have the ability to differentiate into a limited number of cell types, which is controlled by a predetermined level of earlier differentiation. 1.2. Induced pluripotent stem cells (iPSCs) 1.1.2. Origin of isolation Stem cells may also be defined based on their origin. Human embryonic stem cells (ESCs) are harvested from the inner cell mass of the blastocyst of 5-day-old pre-implantation embryos. As ESCs are pluripotent, i.e. can differentiate into the three germ layers, they are a popular tool used in regenerative medicine. Fetal stem cells, unlike ESCs, are isolated later in development, i.e. collected from fetal tissue such as the developing retina (Aftab et al., 2009; Baranov et al., 2014), and are multipotent. Adult stem cells (ASCs), the most developmentally restricted of the 3 cell types, are suggested to be present in small numbers in most major organs. These cells can be multipotent, i.e. give rise to a limited number of cell types specific to the tissue from which they were derived. For example, ASCs can be found in bone marrow, intestinal crypts and in the eye near the limbal region posterior to the cornea (Davanger and Evensen, 1971; Jiang et al., 2002; Snippert et al., 2010). ESCs have played a substantial role in disease modeling and treatment studies in the past decade. However, harvesting ESCs from the inner cell mass of the blastocyst during development restricts their clinical practicality due to limited availability and ethical concerns. The same can be said for fetal cells, which are typically isolated even later in embryonic development. In addition, as ESCs and fetal cells are by definition non-autologous, i.e. not derived from the patient for which they are destined, for cell transplantation an additional obstacle of immune incompatibility exists. For strategies focused on the use of these cell types, life long Shinya Yamanaka and colleagues revolutionized the stem cell field in 2006 when they demonstrated that murine fibroblasts could be reprogrammed into ESC-like pluripotent stem cells, termed induced pluripotent stem cells (iPSCs) (Yamanaka and Takahashi, 2006). They used the knowledge that somatic cell reprogramming can be achieved by transferring a somatic cell's nucleus into an oocyte in order to investigate if the factors needed to maintain ESC identity could perform vital functions in the initiation of pluripotency in somatic cells. Many transcription factors, including Oct3/4, Sox2, and Nanog, work to maintain pluripotency in early embryos (Avilion et al., 2003; Chambers et al., 2003; Mitsui et al., 2003; Nichols et al., 1998). Other genes that are commonly up-regulated in tumors, like Stat3, E-Ras, C-myc, Klf4, and b-catenin, are also known to be involved in the long-term maintenance of ESC pluripotency and the rapid proliferation of ESCs in vitro (Cartwright et al., 2005; Kielman et al., 2002; Matsuda et al., 1999; Phillips et al., 2012). Yamanaka successfully identified four transcription factors, Klf4, Oct3/4, Sox2, and c-Myc (KOSM), which were sufficient for successful reprogramming of mouse embryonic and adult fibroblasts back to a pluripotent state (Fig. 2). Shortly after the original publication, Yamanaka demonstrated that this process could be repeated using human fibroblasts, thus making his work relevant to human disease and an incredibly promising potential resource for cellular transplantation studies (Takahashi et al., 2007). Following these original publications, a plethora of studies focused on the use of different cell types, reprogramming factors and delivery methods were developed (Carey et al., 2009; Gonzalez et al., 2009; Jin et al., 2012; Kim et al., 2009, 2011; Tucker et al., 2013a; Warren et al., 2012; Yoshioka et al., 2013; Yu et al., 2007; Yulin et al., 2012). Such variations were aimed at reducing the need for virally induced genetic insertion of the reprogramming factors, especially the potentially tumorigenic oncogene, c-Myc. Fig. 2. Reprogramming somatic cells into iPSCs. The Yamanaka reprogramming factors (Klf4, Oct4, Sox2, c-Myc) are virally transduced into somatic cells, like murine fibroblasts (pink cells). The reprogramming factors may be packaged in lentiviral vectors, which integrate into the host DNA, or in Sendai viral vectors, which do not require DNA integration. The transduced cells are reseeded onto feeder cells, such as mouse embryonic fibroblasts (MEFs; blue cells), and fed iPSC induction media containing the pluripotency factor, leukemia inhibitory factor (LIF). Colony formation (green cells) will occur approximately one week post-transduction, allowing for the individual colonies to be manually isolated and subsequently expanded into iPSC lines. Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 4 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 The discovery of the iPSC ushered in a new age in the field of regenerative medicine. Unlike ESCs, the human iPSC is not hindered by ethical disputes, and, although not yet fully tested, they should be safer immunologically. That is, a key advantage of the iPSC technology is that these cells can be generated in large numbers using cells taken from the patients for which they are intended. Hence, iPSCs are patient-specific. Additionally, like ESCs, iPSCs are pluripotent and can be differentiated into any cell type of the three embryonic germ layers. The ability to generate patient-specific iPSCs creates novel avenues for disease modeling and transplantation studies. Although the iPSC technology holds great promise, a concern of this technology is that the transgenes used for reprogramming could remain constitutively active or be randomly incorporated into the host genome. Retro- and lentiviral platforms commonly used to deliver reprogramming factors to somatic cells requires DNA to be integrated into the host's genome. Such genomic integration can disturb the normal expression and function of host genes. These concerns have, in large part, been addressed by use of non-integrating delivery systems such as episomal viruses (e.g. Sendai virus), which allow reprogramming factors to be introduced into somatic cells without integrating into the host genome (Ban et al., 2011; Fusaki et al., 2009; Tucker et al., 2013a). Unlike MMLV-based retroviruses or HIV-based lentiviruses, Sendai viruses are RNA-based and function independently of host cell DNA replication. Consequently, transduction with Sendai virus is transient and viral particles can be removed from the infected cells following passage and sub-cloning (Ban et al., 2011; Fusaki et al., 2009). In addition to non-integrating viral delivery systems, several non-viral reprogramming protocols have also been developed. The most promising of these are transient transfection with episomal plasmid DNA (Fontes et al., 2013; Hu and Slukvin, 2013; Okita et al., 2013) and synthetic selfreplicating RNA (Yoshioka et al., 2013). The latter, developed by modifying a noninfectious self-replicating Venezuelan equine encephalitis viral RNA replicon, is a stable DNA-independent single-stranded RNA molecule that was engineered to carry 4 separate reprogramming factors (Yoshioka et al., 2013). Following a single transfection of human fibroblasts efficient generation of integration free iPSCs was achieved (Yoshioka et al., 2013). To give a concise overview of the 3 major categories of stem cells and their advantages and disadvantages from an ophthalmological standpoint, a summary of the above-described points is presented in Table 1. 2. Disease modeling using iPSCs 2.1. Mendelian retinal diseases Being able to offer clinical intervention for inherited genetic disorders is greatly dependent on the understanding of a patient's disease-causing mutations and the consequential pathophysiological mechanism(s) that these disease-causing mutations induce. Lacking the knowledge of a patient's disease-causing gene and how the genetic mutations impact the health and behavior of the affected cells, it would be nearly impossible to generate and provide patient-specific therapeutic strategies. Apart from being a promising cell source for autologous transplantation, a major advantage of the iPSC technology is that it provides the ability to model and study human disease in vitro. In particular, iPSCs are a useful tool for 1) discovery and molecular confirmation of newlyidentified disease-causing mutations; 2) the interrogation of disease pathophysiology in relatively inaccessible tissues such as the retina that cannot be routinely subjected to molecular analysis in living patients; and 3) the rapid testing of novel disease and patient-specific therapeutics, especially important for rare diseases in which animal models that accurately recapitulate the disease phenotype do not exist. As depicted in Fig. 3, the ability to develop a better understanding of disease mechanism will increase the number of therapeutic avenues available to clinicians. The following sections provide some examples of the ways in which iPSCs have been used to model Mendelian eye diseases such as MAK-associated retinitis Table 1 Major stem cell categories and their advantages and disadvantages for development of ocular therapy. Category Embryonic/fetal Adult Induced Derivation/generation Derived from developing embryos (e.g. blastocyst or developing fetus) Derived from developmentally mature organs Generated from terminally differentiated tissue Examples Induced pluripotent stem cell Advantages Able to generate retinal cells, including photoreceptors, RPE, and choroidal endothelial cells Have unlimited self-renewal, so single donations offer enough cells for large number of transplantations No ethical concerns Potential to be patient-specific, less concern for immune rejection Do not form tumors Capable of generating autologous patient-specific retinal cells including photoreceptors, RPE, and choroidal endothelial cells Unlimited capacity for self renewal Less concern of immune rejection No ethical concerns. Ability to recapitulate disease in a dish for study of pathophysiology and testing of therapeutic efficacy Disadvantages Ethical concerns regarding source Not patient-specific Have the potential to form tumors if not properly differentiated and isolated Limited potency Limited capacity for self-renewal Have the potential to form tumors if not properly differentiated and isolated Depending on disease phenotype, genetic correction of patient specific mutations may be required Embryonic stem cells Retinal progenitors Subventricular zone stem cells Photoreceptor precursor cells Mesenchymal stem cell Hematopoietic stem cell Intestinal crypt stem cell Skin derived precursor cell Limbal stem cells Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 5 2.2. MAK-associated retinitis pigmentosa Fig. 3. Schematic depicting how therapeutic modalities available depend on knowledge and progression of retinal degenerative disease in question. A major goal of using patient-specific iPSCs for disease modeling is to acquire more knowledge of the disease's pathogenesis to effectively prevent the retinal degeneration from progressing. The greater the amount of knowledge known about a particular retinal degeneration allows for the development of preventative therapies. For instance, if a disease-causing mutation is known, pre-implantation testing prior to in vitro fertilization could be performed to implant ESCs without the disease-causing mutation. However, corrective treatments are needed as the amount of disease knowledge decreases (green arrow) and disease progression occurs (purple arrow). These avenues start with drug therapies that may slow disease progression. If the genetic cause of a disease is known, gene and/or cell replacement therapy may be a viable option. For late-stage disease, more invasive therapies like optogenetics or retinal prosthesis may be necessary. pigmentosa, Usher Syndrome Type II, CEP290-associated Leber congenital amaurosis, juvenile neuronal ceroid lipofuscinosis, and Best disease, as well as the complex heterogenous disorder, agerelated macular degeneration. Although some inherited retinal diseases are well-understood mechanistically, like ABCA4-associated Stargardt disease, there are still a large number of patients whose disease-causing genes are either unknown or who have known mutations in genes with poorly understood functions. Retinitis pigmentosa (RP) is a particularly devastating type of blinding disease because it gradually claims a person's vision over many years and in some cases also threatens the vision of family members. One of the many obstacles to making gene- and cell-based therapies an everyday reality for people afflicted with RP is the extensive genetic heterogeneity of the disease. To date there are over 60 different genes that have been associated with RP, which can occur in the retina alone or together with other syndromic disorders (Petrs-Silva and Linden, 2014). In our experience, these 60 genes account for less than half of all cases of the disease. That is, over 163 different genes that cause an RP-like phenotype have been identified via genetic sequencing of patients at the University of Iowa Carver Non-profit Genetic Testing Lab (unpublished data). Thus, it is not uncommon for a newly discovered RP gene to cause less than 1% of all cases of the disorder. To make a major impact in the RP field, scientists will need to develop ways to rapidly translate a success achieved in treating one genetic subtype of RP into the treatment of another. That is, they will need a series of interchangeable and reusable reagents and strategies. Recently we used patient-specific iPSC-derived photoreceptor precursor cells generated from patients with retinal degenerative diseases to show that a homozygous Alu insertion, identified in exon 9 of the gene male germ cell-associated kinase (MAK), results in loss of the transcript and an inability to produce functional MAK protein (Tucker et al., 2011b). Loss of MAK was found to disrupt normal photoreceptor cell structure leading to cell death and irreversible blindness. In addition to allowing us to confirm pathogenicity of the MAK mutation, we were also able to identify and confirm the involvement of a unique MAK-specific retinal transcript in disease pathophysiology. This transcript contains an extra 75 base pair exon (exon 12), which is phylogenetically conserved and is only expressed in transcripts that also contain MAK exon 9 (Tucker et al., 2011b). Although mutations in MAK are an uncommon cause of RP in the general population (about 1%) they are quite common among individuals of Ashkenazi Jewish ancestry, accounting for as much as one third of this disease in the Jewish population (Stone et al., 2011). Collectively these findings have enabled progress in the development of gene and autologous cellbased interventions for MAK-associated RP in which patientspecific iPSC-derived photoreceptor cells can be used to confirm therapeutic efficacy in vitro and replace lost cells in vivo. As an additional example of iPSC-derived retinal cells being used to test gene-based therapeutics, a recent study published by Tsang and colleagues show efficacy of AAV8-mediated delivery of Membrane Frizzle-related Protein (MFRP) to rescue the cellular phenotype observed in RPE cells with MFRP-associated RP (Li et al., 2014). 2.3. Usher syndrome type II In a similar study, iPSC-derived photoreceptor precursor cells were used to confirm two disease-causing mutations, one being a novel intronic mutation in the gene USH2A in an adult patient with autosomal recessive RP (Tucker et al., 2013b). In this study, keratinocyte-derived iPSCs were differentiated into multi-layer eyecup-like structures that contained an outer layer of RPE and an inner layer of photoreceptor-like neuronal cells. These photoreceptor precursor cells expressed photoreceptor-specific markers, recoverin and rhodopsin and exhibited axonemes and basal bodies structurally characteristic of photoreceptor outer segments (Tucker Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 6 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 et al., 2013b). Further analysis of the identified USH2A transcripts in these cells revealed that one of the patient's mutations causes exonification of intron 40, a translation frameshift and a premature stop codon. Increased expression of the endoplasmic reticulum (ER) stress-related proteins, GRP78 and GRP94 suggested that the patient's other USH2A variant (a single point mutation resulting in an Arginine to Histidine transition at position 4192) causes disease through protein misfolding and ER stress. To further support the idea that this disease is ER stress-related rather than a developmental defect, photoreceptor precursor cells were transplanted into 4-day-old immunodeficient Crb1/ mice. Transplanted patient-derived cells integrated and formed morphologically and immunohistochemically recognizable photoreceptor cells, suggesting that the mutations in this patient cause degeneration of developmentally normal photoreceptors over time. The role of ER stress in RP-associated photoreceptor cell death is consistent with previous findings in other disease causing mutations. For example, in a similar study Jin and colleagues were able to show that iPSCderived rod photoreceptor precursor cells generated from a patient with autosomal dominant RHODOPSIN-associated RP have elevated levels of ER stress-related proteins and subsequently succumb to apoptotic cell death in vitro (Jin et al., 2011). Unlike MAK-associated RP, neither USH2A nor RHODOPSIN-associated diseases will be amenable to the traditional gene augmentationbased approach (Burnight et al., 2014; MacLaren et al., 2014; Maguire et al., 2009, 2008; Vasireddy et al., 2013). That is, with a coding region of ~19,000 bp, full-length USH2A exceeds the packaging capacity of either of the two viral delivery systems used in the eye to date (i.e. AAV packaging limit is ~4000 bp, lentiviral packaging limit is ~10,000 bp). For this disease, delivery systems with larger carrying capacity could be effective. That said, cloning of mutation-free genes of this size is often problematic. For autosomal dominant diseases such as RHODOPSIN-associated RP, in which the disease-causing mutation causes a gain-of-function, deletion or knockdown of the mutated allele will likely be required. As discussed in detail below, a promising new approach that may be useful for treating both of these classes of disease is CRISPR-based genome editing. 2.4. CEP290-associated Leber congenital amaurosis Leber congenital amaurosis (LCA) refers to a collection of inherited retinal degenerative disorders characterized by nystagmus, little to no recordable electroretinogram, and poor vision presenting from birth (Coppieters et al., 2010). LCA is predominantly inherited in an autosomal recessive manner and of the seventeen loci associated with the disease, the largest contributor is CEP290 (Perrault et al., 2007; Stone, 2007). Thirty percent of LCA patients carry mutations in CEP290, the gene product of which is a cilia-associated protein that functions in photoreceptor outer segment trafficking and ciliogenesis (Craige et al., 2010; Drivas et al., 2013; McEwen et al., 2007; Tsang et al., 2008). Loss of photoreceptor outer segments in patients with CEP290-associated LCA results in severe vision loss. However, some patients retain cone photoreceptors in the central fovea and MRI studies indicated that visual pathways of the inner retina in these patients display normal architecture (Cideciyan et al., 2007). These data, as well as the recent successes garnered in gene augmentation therapy trials for RPE65-associated LCA, support gene and cell therapy as an option to treat CEP290-associated LCA. Patient-specific iPSC-derived photoreceptors provide an excellent opportunity to study therapeutic strategies in vitro. Recently, we demonstrated a ciliogenesis defect in CEP290associated LCA patient cells (Burnight et al., 2014). Previous work indicated that the rd16 mouse model of CEP290-associated LCA exhibited defects in number and length of cilia in serum-starved fibroblasts (Drivas et al., 2013). When we investigated ciliogenesis in CEP290-associated LCA patient fibroblasts with various CEP290 mutations, we observed a decrease in the number of cells forming cilia and in the length of cilia formed. This cilia phenotype was not observed in cells from every CEP290 patient, presumably due to the location and severity of the mutation within the gene. In addition, following gene augmentation, we were able to show that the range of therapeutic dosage was very narrow, i.e. overexpression of wildtype CEP290 itself was toxic. The ability to determine pathophysiology and mutation-specific disease severity in individual patients will give us the opportunity to tailor treatments thereby providing more efficacious outcomes. 2.5. Juvenile neuronal ceroid lipofuscinosis In addition to gene and new gene mutation discovery, iPSCs can be utilized to interrogate disease mechanism, discover potential drug therapies and to evaluate the efficacy of gene correction approaches in human cells. We are currently using iPSCs to gain insight into the devastating autosomal recessive inherited disease, juvenile neuronal ceroid lipofuscinosis, more commonly known as Batten disease. Batten disease is a rare and fatal lysosomal storage disorder that is most often caused by a one-kilobase genomic DNA deletion in the gene, ceroid lipofuscinosis 3 (CLN3) (de los Reyes et al., 2004). Ophthalmologists typically diagnose Batten disease as it presents with severe deficits in central visual acuity due to loss of cone photoreceptors in the macula. Batten disease is usually diagnosed early in a child's life, between the ages of five to seven. Although visual defects and blindness are the first to arise, patients eventually experience seizures, difficulty communicating and walking and severe mental deficit due to extensive central nervous system neuronal cell death. Batten patients face an extremely difficult life of blindness, epilepsy, are often bedridden and usually die during the second or third decade of life. There are currently no treatment options available and little is understood about the molecular role of CLN3 or the mechanism of disease onset when CLN3 is mutated (Seehafer et al., 2011). A key feature of Batten disease is the cytoplasmic accumulation of lysosomal autofluorescent material within neurons (lipofuscin) (Gardiner et al., 1990). We are taking advantage of this easily identifiable phenotypic disease characteristic to discover drug treatment options for Batten patients. We have generated patientderived iPSCs and retinal precursor cells from skin biopsies of human patients with molecularly confirmed CLN3-associated Batten disease. Into these patient-derived cells we are also introducing an apoptosis-dependent reporter construct (Bardet et al., 2008). We are using these diseased patient reporter cells to perform highthroughput drug screens to look for compounds that are capable of mitigating the accumulation of lysosomal lipofuscin deposits and that prevent neuronal apoptosis, thus protecting patient-derived photoreceptor precursor cells from CLN3-associated cell death. Promising compounds can then be further characterized in vivo for their ability to ameliorate lysosomal storage deposits and retinal degeneration in Cln3 knockout mice (Katz et al., 1999; Mitchison et al., 1999). Utilizing autologous stem cells for approaches similar to this one could be a good avenue in which to discover new treatment modalities in addition to providing novel insights into disease mechanisms for previously untreatable diseases. 2.6. Best disease Unlike RP, which is usually caused by mutations in genes expressed in photoreceptor cells, Best disease results from mutations in the RPE-specific gene, BEST1 (Petrukhin et al., 1998). Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 Best disease selectively affects the region of the human retina within one disk diameter of the cone photoreceptor-rich fovea and predisposes affected individuals to the development of vision threatening choroidal neovascular membranes secondary to vitelliform lesions (Kay et al., 2012). Late stages of Best disease are often marked by geographic loss of the RPE underlying the macula and gliotic scarring in the neural retina (Mullins et al., 2007, 2005). Although mutations in BEST1 have long been known to cause Best disease, as mouse models of disease lack the major disease phenotypes, the mechanism by which these mutations cause onset of disease remained unclear. In an elegant study recently published by Singh et al., human iPSC-derived RPE were generated from patients with Best disease and unaffected siblings and used to examine the cellular and molecular disease processes (Singh et al., 2013). They observed that RPE derived from patients with Best disease displayed disrupted fluid flux and increased accumulation of autofluorescent material after long-term feeding of photoreceptor outer segments (Singh et al., 2013). Furthermore, chronic photoreceptor outer segment feeding led to delayed rhodopsin degradation and turnover, cellular responses to calcium were sensitized, and oxidative stress levels were altered (Abramoff et al., 2013; Singh et al., 2013), cell biological findings consistent with altered outer segment-RPE interactions observed in living Best disease patients (Abramoff et al., 2013). Collectively, these findings suggest that BEST1 plays an important role in photoreceptor outer segment turnover and homeostasis and implicates disruption of this pathway in the pathogenesis of Best disease. These studies point to a variety of therapeutic avenues, in particular drug-based studies focused on manipulation of phagocytic and protein degradation pathways may be particularly promising. 2.7. Using iPSCs to model AMD Age-related macular degeneration (AMD) is a genetically heterogeneous and complex disease with over 50 different genetic risk loci identified to date (Gorin, 2012; Liu et al., 2012), 19 of which meet genome wide significance (Fritsche et al., 2013). Early AMD typically presents in the sixth decade of life or later with the formation of lipid-like deposits, called drusen, that accumulate beneath the RPE, as well as drusen-like deposits that appear in the subretinal space (Curcio et al., 2013). A subset of patients with early AMD later develops geographic atrophy of the RPE and underlying choriocapillaris, resulting in the loss of the overlying photoreceptors. Other patients with early AMD develop a complication sometimes referred to as “wet AMD” in which the underlying choroidal vasculature grows into the sub-RPE and/or sub-retinal space forming fenestrated, leaky vascular networks known as choroidal neovascular membranes (CNVs) (Jager et al., 2008). For AMD, all of the therapeutic interventions shown in Fig. 3 could eventually come into play. Drug therapy using the anti-vascular endothelial growth factor (VEGF) agents Avastin and Lucentis have proven very efficacious in ameliorating CNVs, promoting fluid resorption, and slowing vision loss in wet AMD (Agosta et al., 2012; Scott and Bressler, 2013; Stone, 2006). As AMD is genetically heterogenous, traditional gene augmentation approaches seem less promising than for Mendelian diseases. However, cell replacement therapy for patients that have lost photoreceptors, RPE and choroidal (choriocapillaris) endothelial cells is being pursued by a number of groups and, if successful, would be extremely beneficial for thousands of patients worldwide (Kamao et al., 2014; Kokkinaki et al., 2011; Liao et al., 2010; Zhu et al., 2011). In very late stage AMD, in which significant cell loss has occurred, optogenetic and retinal prosthetic approaches may eventually be viable therapeutic options, although the resolution of the latter devices will need to be 7 improved if they are to offer AMD patients a significant improvement in visual acuity. The greatest opportunity for AMD treatment lies in understanding the genetic causes and pathogenic mechanisms thoroughly enough to be able to identify those with a greater propenstity to develop the disease and prevent it from happening through early and efficacious prophylactic intervention. Although the notion that AMD has a strong genetic component was advocated as early as the 1970s (Davanger and Evensen, 1971; Gass, 1973; Jiang et al., 2002; Snippert et al., 2010), our understanding of the biological mechanisms through which specific genetic loci contribute to the development and progression of AMD is still poorly understood. Genome wide association studies of AMD identified two loci on chromosomes 1 and 10 that are highlyassociated with disease risk (Edwards et al., 2005; Haines et al., 2005; Jakobsdottir et al., 2005; Klein et al., 2005; Rivera et al., 2005; Schwartz et al., 2012b; Yamanaka and Takahashi, 2006; Zareparsi et al., 2005). The risk associated with the chromosome 1q locus is predominantly due to a haplotype that harbors a missense mutation (Tyr402His) in the complement factor H (CFH) gene (Avilion et al., 2003; Chambers et al., 2003; Hageman et al., 2006; Mitsui et al., 2003; Nichols et al., 1998; Zhang et al., 2008). The complement cascade was implicated in the pathogenesis of AMD prior to the association of CFH variants with the disease (Bora et al., 2005; Cartwright et al., 2005; Johnson et al., 2001, 2000; Kielman et al., 2002; Matsuda et al., 1999; Mullins et al., 2001, 2000; Phillips et al., 2012), and thus the involvement of this inhibitor in AMD fits into a mechanistic framework of impaired complement regulation and bystander cell injury (Mullins et al., 2011a; Tucker et al., 2011b). In contrast, the mechanism by which the chromosome 10q locus increases AMD risk remains unknown and controversial. The most common high-risk haplotype contains two genes (ARMS2 and HTRA1) with plausible disease-causing mutations. The variants include a non-conservative polymorphism in the ARMS2 gene (Ala69Ser), a complex 144 bp deletion and a 54 bp insertion in the 30 untranslated region of the ARMS2 transcript, and a promoter polymorphism in the HTRA1 gene (Fritsche et al., 2008; Stone et al., 2011). These variants are in strong linkage disequilibrium such that only a few percent of 10q alleles harboring ARMS2 Ala69Ser lack the HTRA1 promoter variant and vice versa. This, coupled with the overall genetic complexity of AMD and a lack of representative animal models, have made it difficult to determine which, if either, of these two genes is responsible for conferring the AMD risk associated with the 10q locus. Recently, Yang and colleagues employed an unbiased proteome screen of N-retinylidene-N-retinyl-ethanolamine (A2E)-aged patient-specific iPSC-derived RPE cell lines derived from patients with high and low risk 10q26 loci (Coppieters et al., 2010; Yang et al., 2014). They reported that cells from patients who were homozygous for the high-risk haplotype had a reduction in superoxide dismutase 2 (SOD2)-mediated antioxidative defense. The authors concluded that the ARMS2/HTRA1 risk alleles decrease SOD2 defense, leaving RPE more susceptible to oxidative damage and thereby contributing to AMD pathogenesis. By elucidating the disease mechanism(s) associated with this locus, new targets for therapeutic intervention have potentially been identified. Historically, the RPE has been seen as the key cell type involved in AMD pathogenesis. Currently, clinical trials focused on stem cellderived RPE cell transplantation are underway. The first, initiated by Robert Lanza and colleagues at Advanced Cell Technology in the UK, was designed to treat patients with dry AMD and Stargardt disease. In this trial patients receive bolus subretinal injections of human embryonic stem cell-derived RPE (NCT01344993). Initial short term reports indicate that transplanted cells attached and Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 8 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 persist within the subretinal space (Schwartz et al., 2012a). Pete Coffey, Dennis Klegg and colleagues have devised a similar trial focused on subretinal transplantation of human ESC-derived RPE cell sheets for the treatment of patients with AMD (NCT01691261). Similarly, in Japan, Masayo Takahashi and collaborators at the RIKEN Center for Developmental Biology have recently transplanted autologous iPSC-derived RPE cell sheets into a woman with AMD (JPRN-UMIN000011929). The latter two studies have focused on sheet transplants rather than bolus cell injection, which have been shown to dramatically improve survivability and integrative capacity (Hu et al., 2012). Cell suspension injections may lead to the formation of isolated cellular clumps that fail to develop into polarized RPE monolayer (Lu et al., 2001; Phillips et al., 2003; Shiragami et al., 1998). To date, the contribution of the choroid, in particular the underlying choriocapillaris vasculature that supports the RPE and photoreceptor cells, has not been thoroughly studied and remains unknown. This lack of knowledge is largely due to the historical inability to clinically image this tissue for better understanding its role in pathogenesis. Recent advances in imaging technology now allow for live imaging, measurement and assessment of choroidal thickness using spectral-domain optical coherence tomography (SD-OCT). Previous studies using the SD-OCT technique on eyes with early-stage AMD without CNVs showed a thinning of the choroid when compared to healthy age-matched controls (Hu et al., 2013; Ko et al., 2013; Sohn et al., 2014). Although it is undeniable that the RPE plays an important role in AMD pathogenesis, findings from our group and others have shown that there is loss of choriocapillaris endothelial cells in eyes with both early- and late-stage AMD (Grunwald et al., 2005; McLeod et al., 2009; Ramrattan et al., 1994). For instance, Sohn and colleagues recently reported that human donor eyes known to have dry AMD are associated with choroidal thinning and that AMD choroids have higher levels of tissue inhibitor of metalloproteinase 3 (TIMP3), which is associated with Sorby's fundus dystrophy, an autosomal-dominant form of macular degeneration (Sohn et al., 2014; Visse and Nagase, 2003). Additionally, a gene set enrichment analysis (GSEA) study on RPE-specific and endothelial cellassociated gene sets found that RPE transcripts were preserved and increased in early AMD, along with a significant decrease of endothelial cell marker expression, suggesting that choroidal endothelial cell dropout is an early event in the pathogenesis of AMD (Whitmore et al., 2013), consistent with morphometric studies showing increased numbers of choriocapillaris ghost vessels with increasing abundance of drusen (Fig. 4A) (Mullins et al., 2011b). These findings indicate that choroidal endothelial cell preservation may slow the progression of or help protect against the onset of early AMD. Stem cell-derived choroidal endothelial cells for the purpose of cellular transplantation may also be required for the reconstruction of the macula in advanced AMD. Although the loss of choriocapillaris EC in early AMD is notable, in endstage AMD (geographic atrophy or choroidal neovascularization) choriocapillaris dropout is profound (Fig. 4B). In advanced AMD, a combined approach in which photoreceptor cells, RPE and choriocapillaris EC are replaced may be necessary in order to restore vision. Endothelial cells can be generated from iPSCs through coculture with primary endothelial cell lines or conditioned media with endothelial cell-specific factors (Choi et al., 2009; Du et al., 2014). For instance, Park et al. successfully generated cord blood iPSC-derived vascular progenitors and used these cells to repair damaged retinal blood vessels (Park et al., 2014b). However, to our knowledge there is no published data showing the differentiation of iPSC-derived choroidal endothelial cells. The choroid is structurally distinct from other vascular tissues, like the brain Fig. 4. Choriocapillaris dropout and membrane attack complex in eyes with macular degeneration. (A) Immunofluorescent labeling of the complement membrane attack complex (green) in a human eye with early/dry AMD. Note the robust labeling at the level of the choriocapillaris (CC) and relative sparing of the RPE. Sections were dual labeled with UEA-I, a marker of viable endothelial cells (red) in the choroid of an eye with dry AMD. Note the abundant “ghost” vessels in the choriocapillaris (asterisks), that are unoccupied by viable endothelial cells. Choriocapillaris loss in eyes with advanced AMD is striking, as depicted in an eye with choroidal neovascularization (B). A comprehensive cell replacement strategy to treat AMD will most likely require addressing the vascular loss in this disease. BlamD: layer of basal laminar deposit, RPE: retinal pigment epithelium, CC: choriocapillaris, CH: outer choroid; RET: neural retina. microvasculature or aortic vasculature, due to its broad, fenestrated and flat lumens with diameters ranging from 20 to 50 mm (Lutty et al., 2010). Nevertheless, the extent to which choroidal endothelial cells are molecularly unique compared to other endothelial cells is not well understood. It is possible that the microenvironment of the choroid causes residing hemangioblasts, precursors of hematopoietic and endothelial cells, to develop and maintain the unique choroidal endothelial cell features. For example, it is thought that VEGF secreted from the overlying RPE is necessary in maintaining the fenestrations seen in the choriocapillaris Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 (Blaauwgeers et al., 1999; Lutty et al., 2010). In order to develop successful treatments for macular degenerations involving choroidal disruption, it is critical to understand if the choroidal endothelial cells vary from other endothelial cells. Therefore, future studies are needed to characterize choroidal endothelial cells to determine if they are unique compared to other endothelial cells at the molecular level. Although the replacement of choroidal ECs derived from iPSCs poses many challenges, there is basis to suspect that autologous EC may be well tolerated in the choroid. IPSC-derived ECs have been shown to integrate with the host vasculature and trigger a relatively modest immune response in a murine model of hindlimb ischemia (de Almeida et al., 2014). Transplanted iPSC-derived ECs may similarly recanalize ghost vessels in the choriocapillaris. This is an active area of investigation. As mentioned above, complement activation has long been shown to play a major role in the pathogenesis of AMD. This notion is further substantiated in that the complement cascade is also active in the choriocapillaris of high-risk CFH AMD eyes (Mullins et al., 2011a). It is possible that complement complexes near the choriocapillaris put stress on the choroidal endothelium and help cause choriocapillaris loss reported in early AMD (Mullins et al., 2011a). The majority of complement membrane attack complexes (MAC) are present in domains surrounding the choriocapillaris e not the RPE or photoreceptors (Fig. 4A) (Mullins et al., 2014). These results further demonstrate the need to elucidate the choroid's role in AMD pathogenesis that could contribute to the development of new treatments for AMD. Development and implementation of efficient choroidal endothelial cell differentiation protocols will empower further studies focused on modeling aspects of AMD specific to the choroid. Stem cell-derived therapeutic modalities are also showing promise for the treatment of another blinding eye disease, glaucoma. The laboratory of Donald Zack has used iPSC-derived ganglion cells to perform high-throughput drug screens for agents that prevent ganglion cell death in glaucomatous cells and for assessing potential neuroprotective growth factors secreted by iPSC-derived retinal ganglion cells (Sluch and Zack, 2014). Also, John Fingert and collaborators recently demonstrated the generation of patientspecific iPSC-derived ganglion cells that have a duplication of the gene, TBK1, which causes normal tension glaucoma. In this study patient-specific iPSC-derived retinal cells were used to show that TBK1 duplication causes increased activation of the autophagy pathway (Tucker et al., 2014). In the following sections, we will review drug, gene replacement, genome editing and cell replacement strategies being employed by our group and others to treat inherited orphan retinal diseases. Included, will be a discussion regarding the debate of eye immune privilege and how donor and autologous stem cell transplantation approaches could affect the recipient eye and immunological homeostasis. 3. Gene augmentation and cell replacement treatment strategies The heritability of Mendelian retinal degenerative diseases, while one of their most frightening features, is also the means by which they will be cured. That is, the fact that these diseases are caused by detectable variations in a gene that is expressed in the retina will make it possible to 1) lessen the risk of recurrence of the diseases in affected families through a combination of genetic testing and genetic counseling, 2) prevent vision loss by early detection of the disease coupled with gene replacement therapy or some other form of treatment (e.g., immune modulation or drug therapy) and 3) create genetically-corrected and immunologically- 9 matched photoreceptor precursor cells that can be transplanted into the retina to restore vision. For numerous diseases, especially those involving significant neurodegeneration or other types of organ failure, drug intervention or gene therapy alone will not suffice. In these conditions, cellular replacement will be required to rescue, refurbish and preserve tissue function. Recessive diseases, particularly those that result in a null phenotype, such as MAK-associated RP and Batten disease, to name two, are often amenable to gene therapy. That is, replacement of the defective mutated gene with the normal transcript prior to photoreceptor cell death could potentially arrest disease progression and prevent subsequent loss of vision. A variety of gene delivery approaches have been developed and used in the eye. These range from non-viral, plasmid transfection approaches, such as cell-penetrating nanoparticles (Read et al., 2010a,b), to viral vector transduction technologies, such as adeno-associated viruses € m et al., 2003; Yang (AAV) (Maguire et al., 2009, 2008; Narfstro et al., 2002) and lentiviruses (Auricchio et al., 2001; Pang et al., 2006). As genetic testing for rare recessive diseases becomes more widespread, patients will be definitively diagnosed earlier in the course of their disease when viral gene replacement therapy has the greatest opportunity to be efficacious, thus sparing the patient's vision. However, for patients with more advanced disease, retinal cell replacement therapy will be necessary. Patient-derived iPSCs have now made this avenue a realistic option. As we have discussed above, patient-derived stem cells harbor disease-causing mutations (Tucker et al., 2013b, 2011b), which make them ideal for disease modeling but a bit less convenient for cell-replacement therapy. That is, for many disorders, the patient's disease-causing mutations must first be corrected prior to transplantation if they are going to be able to have any lasting positive effects. This relative disadvantage is balanced by the tremendous advantage that patient-derived iPSCs are immunologically identical to the patient and thus have the greatest chance of evading the recipient's host immune system with the smallest amount of immunomodulatory medication. 3.1. iPSCs and gene augmentation Gene augmentation is an effective strategy to treat inherited retinal dystrophies, the causes of which are mutations in enzymes or other small genes easily packaged into the well-characterized and efficient adeno-associated virus (AAV) vectors. Numerous clinical trials involving AAV-mediated RPE65 augmentation have restored vision to patients suffering from RPE65-associated LCA e an inherited retinal dystrophy resulting from deficiency of the retinal isomerase, RPE65 (Bainbridge et al., 2008; Hauswirth et al., 2008; Maguire et al., 2009, 2008). Most recently six patients with the X-linked recessive blinding disease choroideremia were given AAV vectors expressing the CHM gene that encodes the Rab escort protein 1 (REP1). Six-months post-injection, increases in visual acuity and retinal sensitivity (measured via dark-adapted microperimetry) were consistent with improved rod and cone function in AAV-treated eyes (MacLaren et al., 2014). We recently demonstrated successful gene transfer to CEP290associated LCA patient iPSC-derived photoreceptor precursors using lentiviral-mediated gene delivery (Burnight et al., 2014). Because CEP290 cDNA (~8 kb) is too large to package in AAV vectors commonly used for clinical gene therapy treatments, we packaged the full-length CEP290 in a lentivirus which can accommodate larger transgenes (8e10 kb packaging capacity) (Balaggan and Ali, 2012). Unfortunately but importantly, we found that overexpression of this large structural protein caused increased cell death in a dose-dependent manner of CEP290-expressing lentiviral vectors (Burnight et al., 2014). Nonetheless, when using a lower Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 10 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 dose, we were able to rescue ciliogenesis in diseased patient cells indicating that restoring full-length CEP290 expression may be an effective treatment option for vision loss in these patients. Due to the adverse cellular toxicity associated with CEP290 overexpression, future experiments in gene transfer will need to address this obstacle. Using cell type-specific promoters whose transcriptional activity is less than that of the strong, viral cytomegalovirus promoter used in our study might decrease the toxicity resulting from CEP290 gene transfer (Burnight et al., 2014). Additionally, one could take advantage of the native CEP290 regulatory elements through genome editing strategies to correct the patient-specific mutation, thereby avoiding ectopic CEP290 expression altogether. 3.2. iPSCs and genome editing Recent advances in genome editing approaches, including the use of zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs), are especially exciting for the potential boost these technologies could provide to cell replacement therapy (Christian et al., 2010; Hockemeyer et al., 2011; Miller et al., 2007; Porteus and Baltimore, 2003). Components of the prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 adaptive immune system can also be used to efficiently and specifically edit human genes using pre-designed 20 nucleotide small guide RNAs (sgRNA) coupled with a human codonoptimized Cas9 nuclease (Cong et al., 2013; Maeder et al., 2013; Ran et al., 2013a,b). Similarly to ZFN- and TALEN-mediated gene correction, cells repair the majority of CRISPR/Cas9-induced double strand breaks (DSBs) by one of two mechanisms. In the absence of a homologous repair template, the cell naturally employs nonhomologous end joining (NHEJ) leading to the insertion or deletion (InDel) of sequence surrounding the DSB (Sun et al., 2012). Repair via NHEJ represents a feasible strategy for intronic mutations in areas of poorly conserved sequence. However, to correct loci at which large deletions or insertions occur or where NHEJmediated InDels would not be tolerated (i.e. splice sites), the sgRNAs and Cas9 must be co-delivered with an engineered repair template containing unmutated, wild-type sequence. DSB repair occurs via homologous recombination using the exogenous repair sequence as template. Homology-directed repair (HDR) in diseased patient cells harboring mutations would presumably restore wildtype gene and protein expression and function. Thus, following CRISPR-based correction, stem cell-derived retinal cells to be used for human transplantation would no longer harbor disease-causing mutations. This technology has the potential to elevate stem cell replacement therapy from being a stopgap, temporary solution, to being a long-term disease remedy. As a proof of principle for the utility of combining genome engineering with the iPSC technology, researchers in the Jaenisch lab combined engineered ZFNs with iPSC technology to create disease and control human iPSC lines to study Parkinson disease (Soldner et al., 2011). Soldner and colleagues designed ZFNs targeting the a-synuclein locus (SNCA), a gene commonly mutated in Parkinson's disease (PD). Using four different targeting strategies, the investigators engineered an array of isogenic and control cell lines that either created two common PD-related mutations (A53T or E46K) in wild-type ESCs or corrected the mutation in PD patientderived iPSCs. Soldner et al. initially employed a drug selection-based strategy to introduce the A53T mutation (a single base pair substitutiondG209Adin exon three of SCNA) into ESCs. The donor construct carried ~600 bp homology on either side of the wild-type sequence at nucleotide 209 (G209) and a floxed puromycinresistance cassette just downstream of the mutation in intron three. Co-electroporation of the ZFN expression plasmids and donor construct into two ESC lines resulted in 3 out of 336 puromycin-resistant clones targeted to the correct locus. Following Cre-mediated excision of the selection cassette and sequence analysis, two of the three clones contained a small deletion in the second allele as a result of the modification. However, the remaining correctly targeted clone maintained pluripotency and was able to differentiate into tyrosine hydroxylase-expressing neurons (Soldner et al., 2011). The second strategy utilized both positive and negative selection to introduce the A53T mutation into both alleles of one of the ESC lines. By incorporating the herpes simplex virus thymidine kinase (HSV-TK) and diphtheria toxin A (DT-A) into the donor cassette, 9 of 41 puromycin- and ganciclovirresistant colonies resulted in correct targeting. One of the clones confirmed by Southern DNA and sequencing analysis contained modification of the second allele. A third strategy generated A53T mutant ESCs without drug selection. The investigators co-delivered ZFNs and a donor plasmid carrying about 1 kb homologous sequence flanking the ZFN cleavage site and the A53T (G209A) point mutation. Additionally, Soldner et al. delivered a reporter cassette expressing eGFP to enrich transfected cells via fluorescence-activated cell sorting (FACS). Analysis of single-cell-derived clones via Southern blotting and PCR-mediated genotyping revealed one of 240 clones was correctly targeted. Cells from the clone maintained pluripotency and were able to differentiate into dopaminergic neurons (Soldner et al., 2011). Instead of using a double-stranded plasmid donor vector, the fourth strategy employed a short single-stranded oligodeoxynuceotide (ssODN) donor construct as a repair template to introduce the PD-associated E46K (G188A) point mutation into ESCs. Using the aforementioned FACS enrichment strategy, the researchers successfully recovered five of 720 single-cell-derived clones with the correct insertion of the E46K/G188A mutation. Lastly, the investigators employed ZFN-mediated genome editing to repair the A53T mutation in PD patient-derived iPSCs. In the absence of drug selection, Soldner et al. delivered ZFNs targeting the G209A mutation in exon three of SNCA and a wild-type sequence-containing donor template carrying ~1 kb homology sequence flanking the targeting site. One of 240 single-cell-derived clones contained the correctly repaired nucleotide at position 209 of SCNA. Subsequent pluripotency marker expression analysis and teratoma formation assays indicated that the genetically corrected clone maintained pluripotency. Moreover, cells from the clone were able to differentiate into tyrosine hydroxylase-expressing dopaminergic neurons that no longer expressed the mutant transcript. This elegant set of experiments demonstrates the utility in combining genome editing strategies with iPSC technology to interrogate disease-causing mutations and therapeutically correct them in vitro. TALENs were used to modify the murine coagulation factor VIII (F8) locus in iPSCs (Park et al., 2014a). Mutations in this locus in humans cause one of the most common bleeding disorders, Hemophilia A. Park et al. engineered TALEN pairs targeted to intron 1 of the murine F8 gene and electroporated plasmids encoding the most efficient pair into wild-type iPSCs. After clonal selection and PCR analysis, the researchers recovered six of 432 (1.4%) colonies with a 140 kbp inversion resulting from non-allelic homologous recombination (HR), thus creating model cell lines containing a common mutation in patients with Hemophilia A. Expression analysis in the inversion clones indicated loss of F8 mRNA and protein. Importantly, Park and colleagues were able to restore F8 expression through inducing reversion of the 140 kbp segment by delivering the same TALEN pair to the mutant clones (Park et al., 2014a). Thus, the researchers demonstrated the use of genome Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 editing technologies to both model and correct genetic diseases in the culture dish. Recently, Wu and colleagues utilized the CRISPR-Cas9 system to correct a dominant mutation in the murine Crygc gene e mutations in which cause cataracts (Wu et al., 2013). Co-injection of Cas9 mRNA and an sgRNA targeting the mutant allele with one of two oligonucleotide repair templates into Crygc mutant embryos yielded HDR-mediated repair in 5 of 29 (Oligo-1; 17.2%) and 4 of 27 (Oligo-2; 14.8%) live-born pups. All mice that carried the corrected allele induced by HDR were free of cataracts and the lenses were histologically normal compared to non-rescued mutant mice (Wu et al., 2013). These results demonstrated that the CRISPR-Cas9 system could be used to correct genetic disease phenotypes in vivo. While the combination of genome editing strategies with iPSC technology represents a powerful avenue by which to investigate the pathophysiology of inherited retinal dystrophies as well as efficacy of treatment, there is concern that DSB induction at off-target sites will create adverse effects such as genotoxicity. Indeed, the CRISPR/Cas9 system can tolerate up to five mismatches in the sgRNA target site (Fu et al., 2013; Hsu et al., 2013; Pattanayak et al., 2013). In addition to determining efficacy of therapeutic strategies, evaluation of undesirable off-target modifications will be essential when employing genome editing in the clinic. The majority of bioinformatically predicted off-target modifications can be assessed through PCR amplification and Surveyor nuclease digestion (Fig. 5). Surveyor nuclease is sensitive enough to detect from about three percent modifications at a locus (Qiu et al., 2004), however, genome wide analysis using deep sequencing technologies allows for thorough assessment of safety in modified cells. Several groups have performed off-target analyses using these methods and results indicate that modification at unintended loci occurs very rarely (Duan et al., 2014; Güell et al., 2014; Li et al., 2013; Soldner et al., 2011; Wang et al., 2013; Wu et al., 2013). In addition, Wu and colleagues evaluated off-target mutations in CRISPR-Cas9 modified mice via DNA sequencing of PCR products from ten potential off-target sites in twelve mice. No mutations Fig. 5. Off-target mutation analysis in cells treated with CRISPR/Cas. A) Schematic representation of the EMX1 locus. A previously described sgRNA targeting just upstream of exon 3 (red arrow) of EMX1 (sg5) (Ran et al., 2013a) was delivered with the wild-type Cas9 nuclease to HEK293T cells. B) The Optimized CRISPR Design tool (http://crispr.mit.edu) was used to bioinformatically predict the off-target sites (Cong et al., 2013). The top sites are shown in the table. Bolded letters indicate base changes from the sgRNA, sg5. C) Surveyor nuclease activity at the on-target (sg5) and four off-target (OT) sites in duplicate. Genomic DNA from cells treated with sg5 (þ) or untreated controls () were subjected to Surveyor nuclease detection analysis (Ran et al., 2013b). sgRNA: small guide RNA, PAM: protospacer adjacent motif, OT: off-target. 11 were recovered in ten of the twelve mice evaluated. Of the remaining two, only one site carried off-target modifications (Wu et al., 2013). To address the issue of undesirable off-target modifications, investigators employ a modified Cas9 nuclease (Ran et al., 2013a). The modified Cas9 carries a catalytic amino acid substitution (D10A) in the conserved RuvC nuclease domain converting the enzyme to a ‘nickase’, i.e. Cas9 cleaves only the strand noncomplementary to the guide RNA. Single-stranded nicks are repaired by the base excision repair pathway (BER) (Dianov and Hübscher, 2013), and thus maintain genome integrity. By delivering the Cas9-nickase guided by a pair of sgRNAs targeting opposite strands of the target locus, the nicking enzyme creates DSBs with minimal off-target modification. Ran and colleagues recently demonstrated a 50e1500-fold reduction in off-target cleavage using this approach. Importantly, employing the nickase strategy maintains on-target activity (Ran et al., 2013a). Similarly, shortening the sgRNAs can reduce off-target activity. Fu et al. showed that sgRNAs of 17 or 19 nucleotides in length reduced off-target activity by as much as 5000-fold in some cases (Fu et al., 2014). Again, these truncated guides showed comparable on-target activity. Employing either truncated guides or paired nickases, or both strategies in concert can greatly reduce undesired off-target effects. These exciting technologies, in addition to future developments in genome editing, will be essential in allowing immunologicallymatched and genetically wild-type cell replacement in heritable forms of blindness. 3.3. iPSCs for testing of therapeutic efficacy Although human stem cells represent an exciting new frontier for treatment of inherited blinding diseases, a sobering aspect of this otherwise exciting progress is the sluggish pace at which the few therapies that are available today have moved from “proof-ofconcept” stages in animals to a fully-approved treatment available to patients who need them. An example of this is the substantial amount of time it took for RPE65 gene replacement therapy to become a reality for patients afflicted with RPE65-associated LCA. Efficacy of RPE65 gene replacement was demonstrated in a canine model in 2001 (Acland et al., 2001), and over thirteen years later, fewer than 100 patients have been treated world-wide and the Phase III clinical trial is still ongoing. This raises the possibility that some diseases might be so rare in the population that it is not economically logical and feasible to bring a treatment through the regulatory gauntlet and into clinical availability. One possible solution for disorders that are so rare that they are below the commercial viability economic threshold is to use autologous patient-derived cells to test the molecular efficacy of a drug or viral-mediated gene therapy. Although the optimal dosages of therapies may not be identical in culture and in vivo, demonstrating efficacy and a range of tolerated and effective doses in patient-specific cells will be an important step forward. We envision that the identified therapies can then be delivered to one eye in a compassionate use manner. Patients treated in this way can be monitored at intervals using conventional clinical measures such as visual acuity, Goldmann perimetry, ganzfeld electroretinography and optical coherence tomography. In the event treated eyes fare worse than untreated ones, the compassionate use treatment can be stopped until the reason for the poorer outcome can be identified and an improved treatment can be developed. When treated eyes display noticeable improvement in vision than untreated eyes over time, the treatment can then be offered to new patients and the second eyes of the initial patients can also receive treatment. In adopting a treatment approach like this, a series of increasingly predictable, reusable Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 12 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 parts can be assembled and used to devise a compassionate use therapy for retinal disorders, regardless of their rarity. cells will enhance the efficiency of drug discovery and decrease the time lag between compound discovery and Phase I clinical trials. 3.4. iPSCs and drug discovery 3.5. Retinal transplantation and cellular replacement For translational applications like discovering novel pharmacotherapeutics, human stem cell-based assays represent a physiologically relevant and high-throughput means to assess efficacy and toxicity early on in the drug development pipeline. Human stem cells are a means for expediting early proof-of-concept studies for potential drug candidates. As indicated above, retinal cells derived from iPSCs are faithful representatives of their in vivo counterparts (Tucker et al., 2013b). High-throughput drug screens of differentiated iPSC-derived cells have been suggested as a means for drug discovery and personalized medicine (Desbordes and Studer, 2013; Inoue and Yamanaka, 2011). Drug screening using extra-ocular iPSC-derived cells from patients with rare diseases has shown excellent promise. Screens have been performed on iPSC-derived cardiomyocytes (Cerignoli et al., 2012; Mordwinkin et al., 2013), forebrain neurons (Luo et al., 2013; Yahata et al., 2011), motor neurons (Egawa et al., 2012), and hepatocytes (Kim et al., 2013), to name a few examples. Combining the power of differentiated ocular cells with highthroughput screens will likely prove effective for discovering new pharmacologic treatments for delaying the progression of retinal degenerative diseases. High-throughput drug screens will need to be highly organized and well thought out, however, if they are to successfully identify reliable drug treatment candidates. Screening assays will need to be multiplexed to simultaneously assess the proliferation, morphology, differentiation state, viability and function of specific iPSC-derived retinal cell types that would enable the monitoring and testing of disease-relevant phenotypes (Wright et al., 2013). To date there have been a limited number of reports looking at the effects of drugs on iPSC-derived retinal cells. David Gamm and collaborators reported the restoration of ornithine aminotransferase activity in iPSC-derived RPE from a patient with gyrate atrophy following treatment with vitamin B6 (Meyer et al., 2011a). It has been known for some time that vitamin B6 is beneficial for patients with gyrate atrophy. However, the patient of interest in this study was previously unresponsive to vitamin B6 supplementation. This study highlighted the use of iPSC-derived RPE to test the specific cellular population targeted in gyrate atrophy to more accurately test vitamin B6 efficacy. In another study, the lab of Masayo Takahashi generated patientspecific iPSC-derived rod photoreceptor-like cells harboring mutations in several genes that cause retinitis pigmentosa like RP1, PRPH2, RHODOPSIN, and RP9 (Jin et al., 2011). This study showed that diseased RHODOPSIN-positive iPSC-derived retinal cells perished after 120e150 days in the culture dish. The authors then treated cells with 3 antioxidant vitamins, a-tocopherol, ascorbic acid and b-carotene. They observed that cells treated with atocopherol led to a statistically significant increase in survival of diseased iPSC-derived photoreceptor lines, providing proof-ofprincipal for drug screening of iPSC-derived retinal cells. IPSC technology offers an unlimited source of disease- and patient-specific material for automated, high-throughput drug screening systems to screen libraries of drug compounds. As evidence for the efficacy of this strategy, work from the laboratory of Donald Zack, which utilized a high-throughput RNA interference screen and primary mouse retinal ganglion cells, demonstrated that large compound libraries could be effectively screened in a rapid fashion to identify novel therapeutics that promote retinal ganglion cell survival (Welsbie et al., 2013). Ultimately, replacing primary neurons in these experiments with iPSC-derived retinal Cell replacement therapy is simply defined as replacing dead or damaged cells with healthy cells to restore the function that a specific cell population provides within a tissue. The retina is a good candidate for cell replacement therapies for several reasons. Compared to other constituents of the central nervous system (CNS), the posterior retina is very accessible to therapeutic delivery through subretinal injection, a standard technique that can be performed by most vitreoretinal surgeons. Clinical diagnosis and post-intervention examining of the retina are also feasible compared to other regions of the CNS due to the transparency of the cornea, lens, and intraocular media. However, there is a longstanding argument amongst clinicians and vision scientists pertaining to the following key questions: 1) What is the optimal stem cell source for production of outer retinal cells?; 2) What is the optimal stage of differentiation for posttransplant survival, integration and cellular function?; and 3) How will the local diseased environment respond to transplanted cells immunologically? 1) What is the optimal stem cell source for production of outer retinal cells? Retinal progenitor cells (RPCs) have been investigated as a potential option for retinal cell replacement therapy. These cells can be isolated from early embryonic tissue and give rise to ganglion, amacrine, horizontal, retinal pigmented epithelium, and photoreceptor cells (Baranov et al., 2014; Chatoo et al., 2010; Hafler et al., 2012; Jadhav et al., 2006; Luo et al., 2014; Rompani and Cepko, 2008; Rowan et al., 2004; Schmitt et al., 2009). Subretinal transplantation of GFP-positive RPCs into retinal degenerative mouse models results in migration of the transplanted cells into the outer nuclear layer, differentiation into immunohistochemically identifiable rod photoreceptor cells and functional rescue of blindness in the form of improved pupillary light responses (Klassen et al., 2004). Likewise, transplantation of human RPCs into the subretinal space of the RCS rat was shown to slow disease progression (Luo et al., 2014). As promising as RPCs are, as indicated above, these cells are obtained from post-mortem embryonic globes and as such are limited in number, rife with ethical concerns and are immunologically dissimilar from the transplant recipient. In light of this, the two most promising stem cell types for human retinal transplantation are human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). As indicated above, ESCs are harvested from developing embryos approximately 4e5 days after fertilization just prior to implantation (Thomson et al., 1998), whereas iPSCs can be generated by reprogramming various cell types with cocktails of transcription factors, such as adult dermal fibroblasts via viral transduction of the transcription factors Oct4, Sox2, Klf4, and c-Myc. Both ESCs and iPSCs can be expanded to yield sufficient cell numbers for clinical applications and both have the capacity to generate all of the various cell types of the retina. To date, differentiation protocols capable of generating photoreceptor precursor cells from both ESCs and iPSCs have been developed (Ikeda et al., 2005; Lamba et al., 2006; Meyer et al., 2011b, 2009; Nakano et al., 2012; Osakada et al., 2008, 2009a,b; Tucker et al., 2013a, 2011a). Like RPCs, ESCs and iPSCs each possess their own pros and cons. For example, ESCs could represent a ‘universal’ donor stem cell population, but the harvesting and use of ESCs is highly controversial from an ethical point of view, and like RPCs, ESCs would be an allogenic transplanted cell population, as donor and recipient would not be immunologically matched. This situation could have Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 negative consequences within the retina following transplantation, including but not limited to, rejection of the transplanted cells and an unwanted inflammatory response in the eye. On the other hand, iPSCs represent an autologous donor population because they are generated from somatic cells isolated from the patient for which they are intended (e.g. skin-derived fibroblasts or keratinocytes). However, as we will describe further below, the retinal immune environment may be more complicated, and even this cell type may require post-transplant immune modulation. A drawback to the use of autologous iPSCs is that since they are generated on a patientspecific basis, they will take much longer to produce and as they will be produced ‘as needed’ they will require significantly more manpower and resources to meet clinical demands. Additionally, depending on the patients' original disease-causing mutation, genetic correction may also be required. Although time will tell which of these cell types will be optimal for therapeutic cell replacement, we have chosen to hedge our bets with the autologous iPSC-based strategy. 2) What is the optimal stage of differentiation for post-transplant survival, integration and cellular function? The next big question after deciding which stem cell source to utilize is at what stage of differentiation should cells be in order to achieve optimal levels of post-transplant survival, integration and function? For photoreceptor cell transplantation, the post-mitotic photoreceptor precursor cell has been shown to be at the optimal stage of development for retinal transplantation (i.e. it possesses the greatest capacity for cellular integration and restoration of retinal structure) (MacLaren et al., 2006). Unfortunately, like RPCs, in humans these cells are isolated during late embryonic development, which raises serious ethical and practical concerns (i.e. it would be difficult to obtain sufficient numbers of viable cells for transplantation). Fortunately, both ESCs and iPSCs have all been shown to give rise to photoreceptor precursor cells postdifferentiation and following transplantation from new functional photoreceptors (Bartsch et al., 2008; Gonzalez-Cordero et al., 2013; Lamba et al., 2009; Tucker et al., 2013b, 2011a; West et al., 2012; Yao et al., 2011). Of the differentiation paradigms used, ours was developed by combining aspects of previously published differentiation strategies (Ikeda et al., 2005; Lamba et al., 2006; Osakada et al., 2008) which take into account the role of bone morphogenic protein (BMP) and Wnt signaling pathway inhibition in neuroectodermal development (Anderson et al., 2002; Lamb et al., 1993; Mukhopadhyay et al., 2001), the role of IGF-1 in anterior neural/eye field development (Pera et al., 2001), and Notch pathway inhibition in photoreceptor development (Jadhav et al., 2006). Using this differentiation program, iPSCs readily form embryoid bodies within days and early retinal progenitor cells 13 (RPCs) within weeks. These iPSC-derived RPCs express the retinalspecific markers OTX2, SIX6, VSX2, RX and CRX. Following 2e3 more months, these cells further differentiate into eyecup like structures with post-mitotic photoreceptor precursor cells that express the photoreceptor-specific markers recoverin and rhodopsin (Fig. 6), phosducin, red/green-opsin and blue-opsin (Tucker et al., 2013a,b, 2011a,b). Although promising for photoreceptor-only diseases, the optimal developmental target is yet to be determined for RPE and choroidal endothelial cells. As such, in situations where multiple cell types are required further experimentation is needed. In disease states such as these, autologous cell sources will likely be critical. Regardless of stem cell source and differentiation stage, differentiated retinal cell types will need to be purified from other cell types present in the culture before transplantation in the clinic. Generating and purifying photoreceptors or choroidal endothelial cells in great enough numbers presents a challenge. Lamba and colleagues purified photoreceptors generated from human iPS cells using fluorescence activated cell sorting (FACS) after transduction with a lentivirus expressing GFP from a photoreceptor-specific regulatory elements. The purified photoreceptors integrated into mouse retina and expressed photoreceptor-specific markers Otx2, recoverin, and rhodopsin, supporting the proof-of-principle that differentiated photoreceptor precursors can be purified and transplanted successfully (Lamba et al., 2010). Genetically modified photoreceptors, however, would not be desirable for treatment, thus alternate methods of purification should be explored. Lakowski et al. isolated photoreceptor precursor cells from developing murine retinae using the photoreceptor cell surface antigens Cd24a and Nt5e and FACS. The researchers transplanted the selected photoreceptor precursor cell population into wild-type or degenerative murine host eyes (Crb1/ or Prph2/). The selected cells migrated into the outer nuclear layer, acquired photoreceptor morphology and expressed outer segment markers at an 18-fold higher integration efficiency than that of unsorted cells (Lakowski et al., 2011). These important experiments indicate the feasibility of using cell selection strategies employing antibodies recognizing cell surface markers to purify photoreceptors for transplantation. One can envision a similar strategy for use with choroidal endothelial cells. 3) How will the local diseased environment respond to transplanted cells immunologically? 3.6. Retinal immune landscape The eye has long been believed to be an organ that boasts “immune privilege” from the body's surrounding immune system. Fig. 6. IPSC-derived photoreceptors express photoreceptor-specific markers. A) Immunofluorescent labeling of recoverin (green), tubulin (red) and DAPI (blue) in differentiated iPSC-derived photoreceptor cultures. Inset shows a magnified view of recoverin-positive photoreceptor-like cells. B) High magnification example of a differentiated neural rosette that is comprised of photoreceptor-like cells that express the photoreceptor-specific markers, recoverin (green) and rhodopsin (red). Scale bar ¼ 400 mm. Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 14 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 It is believed that this “immune privilege” exists for the purpose of protection from unwanted inflammatory responses and helps to maintain the intraocular microenvironment. The first description of “ocular immune privilege” was made by Dutch ophthalmologist J.C. van Dooremaals in 1873 (Van Dooremaal, 1873). Dr. van Dooremaals observed that tumor cells injected into the anterior chamber of the eye survived, proliferated and formed intraocular tumors (Van Dooremaal, 1873). Many years later it was demonstrated that the eye is not entirely “immune privileged,” but the response to intraocular antigens is highly controlled and specialized, a phenomenon that became known as anterior chamber-associated immune deviation (ACAID) (Kaplan and Streilein, 1977; Stein-Streilein and Streilein, 2002; Streilein, 1987; Streilein and Niederkorn, 1981). ACAID is the process by which the eye communicates with the surrounding immune system via the spleen to tolerate antigen presenting cells in the eye and thus avoid a delayed, heightened systemic immune response (Stein-Streilein and Streilein, 2002; Streilein and Niederkorn, 1981). A common clinical example of the tolerance allowed due to ACAID is the allogenic transplantation of avascular corneal grafts, a highly successful surgery that requires no immunological matching between donor and recipient, merely local immunomodulation via topical drops. Although the retina does not possess the same level of immune privilege as the anterior chamber, the blood-retinal barrier (BRB) affords some protection. The BRB acts as a tight barrier between the retina and that of the systemic circulation, namely circulating immunomodulatory and effector leukocytes. The BRB actually consists of two separate anatomical sites: 1) the endothelial cells, pericytes, and glia of the non-fenestrated inner retinal vasculature and 2) the tight junctioncontaining, monolayered RPE on Bruch's membrane between the fenestrated choroidal vasculature and outer retinal vasculature (Crane and Liversidge, 2008). In a normal, healthy retina, though not immune privileged, the BRB provides the retina with an immune advantage, keeping it isolated from the surrounding immune system. However, when the retina is damaged, particularly if the integrity of the RPE and choroidal vasculature is compromised, circulating immunomodulatory factors have easier access to the neural retina. In most degenerative retinal diseases, the RPE is significantly injured. Using human donor eyes from patients with various retinal degenerative diseases like ABCA4-associated Stargardt disease, Best disease and AMD, we have shown that loss of RPE is a common characteristic among these disorders (Mullins et al., 2012, 2007). Support for the BRB playing a major role in conferring protection from the systemic immune system lies in evidence that the subretinal space becomes much more proinflammatory in the face of photoreceptor and RPE degeneration (Chinnery et al., 2012; Mullins et al., 2012; Rutar et al., 2010). There are also reports that inflammation is heavily involved in the pathogenesis of AMD (Ambati et al., 2013; Anand et al., 2003; Tarallo et al., 2012; Whitcup et al., 2013). Furthermore, it has been suggested that the immune system may be critical in the pathogenesis of Batten disease. Cellular infiltrates have been observed in the vitreous cavity of molecularly-confirmed Batten patients (Fig. 7), and these patients also possess circulating autoantibodies, particularly directed against GAD65 (Chattopadhyay et al., 2002; Drack et al., 2014; Pearce et al., 2004). Immunosuppressive agents have demonstrated efficacy in slowing the progression of CLN3-associated Batten disease in a genetic mouse model and a human patient (Drack et al., 2014; Seehafer et al., 2011). The above examples of how loss of BRB integrity in numerous retinal degenerative diseases leaves the retina vulnerable to circulating immune cells and immunomodulatory factors are the reason we believe that the most promising cellular population for future use in human cellular transplantation trials is that of autologous, immunologically-matched patient-derived iPSCs. While ESCs may be more readily available in a shorter timeframe, the fact that they are not immunologically matched between donor and recipient makes it highly likely that they will incite an acute immune response, leading to rejection of the transplanted cells and perhaps further damage to an already diseased eye. This concern is of particular importance if the patient receiving ESCs still has some functional vision remaining. That being said, ESCs would likely still be a good option for transplantation and would be tolerated to a better degree in early-stage diseased retinas in which the RPE is relatively undamaged and BRB integrity remains intact. While using autologous iPSCs will likely reduce the incidence of immunological reactions and rejection of transplanted cells, the degree to which immunologic matching will be required for the survival, integration, function, and longevity of stem cell-based retinal transplants is a critical unanswered question in the field. Definitively testing this hypothesis will allow us to conserve societal resources (if patient-specific cell sources prove not to be required for successful therapeutic transplantation); or, prevent harmful immune activation in visually impaired patients (if our studies show that immune matching of the transplant is necessary). 4. Summary Patient-specific induced pluripotent stem cells have emerged as a promising tool for identification and interrogation of diseasecausing mutations, testing efficacy of novel therapeutics, and as a cell source for autologous retinal cell replacement. As depicted in Fig. 8, the key to patient-specific therapeutic development is thorough phenotypic assessment and accurate clinical diagnosis of Fig. 7. Cellular infiltrates in the vitreous cavity of a patient with CLN3-associated Batten disease. A) Fundus photograph of the right eye of a child with molecularly-confirmed CLN3associated Batten disease showing bulls eye maculopathy and granularity of the fovea, both common presentations in eyes afflicted with Batten disease. B) Slit lamp photograph of the same eye in (A) and a magnified view of cellular infiltrates present throughout the vitreous cavity (outlined in red box). Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 15 Fig. 8. Schematic diagram depicting the strategy for use of iPSCs for the interrogation and development of patient-specific therapeutics for retinal degenerative diseases. inherited retinal disease, taking into account patient and family history, high-resolution retinal imaging (OCT and fundoscopy), visual field and electroretinographic analysis, followed by molecular confirmation (genotyping) of disease-causing mutations via sequencing of patient DNA (from blood). A skin biopsy obtained during the initial clinical visit can be used for generation of autologous patient-derived iPSCs. IPSCs and iPSC-derived retinal cells can then be used to interrogate disease pathophysiology, develop drug, genome editing, gene augmentation and cell-based therapies, as well as to discover novel mutations that cannot be identified using standard sequencing methods, such as those located in promoter or intronic regions of the gene. Finally, strategies developed and tested against patient-specific disease mutations and phenotypes can then be utilized for human gene and cell replacement therapies. 5. Future directions Many exciting developments in the field of iPSC technology present an excellent opportunity to treat inherited retinal degenerative diseases and ultimately improve patients' lives. IPSCs can be employed to investigate and determine disease mechanisms from individual patients as well as determining pathogenicity of novel mutations. Knowledge gained from these experiments can be used Please cite this article in press as: Wiley, L.A., et al., Patient-specific induced pluripotent stem cells (iPSCs) for the study and treatment of retinal degenerative diseases, Progress in Retinal and Eye Research (2014), http://dx.doi.org/10.1016/j.preteyeres.2014.10.002 16 L.A. Wiley et al. / Progress in Retinal and Eye Research xxx (2014) 1e21 to develop and test drug- and gene-based therapies. Transplantation of gene-corrected patient-specific iPSC-derived retinal neurons will treat individuals with advanced-stage retinal degenerative disease. These approaches will be expedited through production of clinical-grade reagents (i.e. patient-specific iPSC-derived retinal photoreceptor cell lines and viral vectors) in dedicated current good manufacturing production (cGMP) facilities. Clinicalgrade stem cells and therapeutic vectors can be utilized in a compassionate use manner to treat aggressive neurodegenerative blinding diseases to pave the way for treatments of other rare inherited retinal dystrophies that fall below the commercial viability threshold. For patients with vision threatening diseases, for the first time there is cause for real hope. Acknowledgments We would like to graciously thank the following funding organizations: NIH Directors New Innovator Award 1-DP2-OD00748301; NEI EY017451; HHMI; Foundation Fighting Blindness; Stephen A. Wynn Foundation; Grousbeck Family Foundation; Leo, Jacques & Marion Hauser Family Vision Restoration Fund; NIH F32 EY022834. References Abramoff, M.D., Mullins, R.F., Lee, K., Hoffmann, J.M., Sonka, M., Critser, D.B., Stasheff, S.F., Stone, E.M., 2013. Human photoreceptor outer segments shorten during light adaptation. Invest. Ophthalmol. Vis. Sci. 54, 3721e3728. http:// dx.doi.org/10.1167/iovs.13-11812. 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