Wiley et al PRER

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Progress in Retinal and Eye Research 44 (2015) 15e35

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Progress in Retinal and Eye Research

journal homepage: www.elsevier .com/locate/prer

Patient-specific induced pluripotent stem cells (iPSCs) for the studyand 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 Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences, University of Iowa, Iowa City, IA, USAb Howard Hughes Medical Institute, University of Iowa, Iowa City, IA, USA

a r t i c l e i n f o

Article history:Received 31 July 2014Received in revised form15 October 2014Accepted 16 October 2014Available online 4 November 2014

Keywords:Induced pluripotent stem cellsRetinal degenerationGene therapyCRISPR-based genome editingCell transplantation

List of abbreviations: AMD, age-related macular demented epithelium; ESC, embryonic stem cell; ASC, apluripotent stem cell; RP, retinitis pigmentosa; AAV,Leber congenital amaurosis; TALEN, transcription actCRISPR, clustered regulatory interspaced short palindrnuclease; sgRNA, small guide RNA; DSB, double-strangous end joining; InDel, insertion or deletion; HDR, horetinal progenitor cell; ACAID, anterior chamber-asso* Corresponding author. Stephen A. Wynn Institut

College of Medicine, University of Iowa, DepartmentSciences, 375 Newton Road, Iowa City, IA 52242, USA

E-mail address: [email protected] (B.A. Tuc1 Percentage of work contributed by each autho

manuscript is as follows: Luke A. Wiley: 20%; ErinSongstad: 20%; Arlene V. Drack: 7.5%; Robert F. MullinBudd A. Tucker: 17.5%.

2 These authors contributed equally to this manusc

http://dx.doi.org/10.1016/j.preteyeres.2014.10.0021350-9462/© 2014 Published by Elsevier Ltd.

a b s t r a c t

Vision is the sense that we use to navigate the world around us. Thus it is not surprising that blindness isone of people's most feared maladies. Heritable diseases of the retina, such as age-related maculardegeneration 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 withretinal 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 anddevelop various treatment modalities for the treatment of inherited retinal degenerative disease. We willfocus on the use of iPSCs for interrogation of disease pathophysiology, analysis of drug and gene ther-apeutics and as a source of autologous cells for cell transplantation and replacement.

© 2014 Published by Elsevier Ltd.

Contents

1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 161.1. Stem cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16

1.1.1. Cellular potency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 171.1.2. Origin of isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17

1.2. Induced pluripotent stem cells (iPSCs) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 172. Disease modeling using iPSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 18

2.1. Mendelian retinal diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 182.2. MAK-associated retinitis pigmentosa . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 192.3. Usher syndrome type II . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 19

generation; RPE, retinal pig-dult stem cell; iPSC, inducedadeno-associated virus; LCA,ivator-like effector nuclease;omic repeat; ZFN, zinc fingerd break; NHEJ, non-homolo-mology-directed repair; RPC,ciated immune deviation.e for Vision Research, Carverof Ophthalmology and Visual. Tel.: þ1 319 355 7242.ker).r in the production of theR. Burnight: 20%; Allison E.s: 7.5%; Edwin M. Stone: 7.5%;

ript.

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L.A. Wiley et al. / Progress in Retinal and Eye Research 44 (2015) 15e3516

2.4. CEP290-associated Leber congenital amaurosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.5. Juvenile neuronal ceroid lipofuscinosis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.6. Best disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202.7. Using iPSCs to model AMD . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 21

3. Gene augmentation and cell replacement treatment strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.1. iPSCs and gene augmentation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 233.2. iPSCs and genome editing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243.3. iPSCs for testing of therapeutic efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 253.4. iPSCs and drug discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.5. Retinal transplantation and cellular replacement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 263.6. Retinal immune landscape . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

4. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 285. Future directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

Acknowledgments . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

Fig. 1. The epigenetic landscape of somatic cells. Conrad Waddington proposed thatpluripotent stem cells terminally differentiate into somatic cells. It is now known thatpluripotent stem cells undergo stepwise differentiation (blue arrows), producinglineage-specific progenitor cells that go on to further differentiate into germ layer-specific progenitor cells (EndoSP, MesoSP and EctoSP). These layer-specific progeni-tor 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 cellresearch pioneers, like Gurdon and Yamanaka, demonstrated that somatic cells fromany germ layer could be reprogrammed into pluripotent stem cells (green arrow) anddirectly converted from one somatic cell type into another somatic cell type (purplearrow). PS: pluripotent stem cell, LSP: lineage-specific progenitor, EndoSP: endoderm-specific progenitor, MesoSP: mesoderm-specific progenitor, EctoSP: ectoderm-specificprogenitor.

1. Introduction

Heritable retinal degenerative diseases, which include Mende-lian disorders such as retinitis pigmentosa, Stargardt disease andLeber congenital amaurosis, as well as the more complex andheterogeneous disease, age-related macular degeneration (AMD),are collectively the leading cause of incurable blindness in thedeveloped world (Huang et al., 2011). For years, ophthalmologistsand vision scientists have dreamed of restoring vision to those thathave lost this precious sense. Fortunately, despite widespreaddeath of the photoreceptors, retinal pigmented epithelium (RPE)and choroidal endothelial cells ewhich comprise the outer retina–the inner retina, which consists of ganglion cells, bipolar cells,amacrine cells, horizontal cells, and Müller glia is largely spared inthese diseases (Huang et al., 2011; Mullins et al., 2009, 2012).Preservation of the inner neural retina presents an opportunisticenvironment inwhich preservation and replacement of these outerretinal cell populations could maintain and restore visual function.

Over the past decade, studies employing stem cells for diseasemodeling and treatment of incurable diseases have gained mo-mentum in the field of regenerative medicine. The groundwork ofthe stem cell field goes back more than 50 years when Sir JohnGurdon demonstrated that nuclear transfer of tadpole nuclei into arecipient oocyte resulted in the generation of clonally identicalfrogs (Gurdon, 1962). In contradiction to the concept classicallyillustrated by Conrad Waddington's proposal of the ‘epigeneticlandscape,’ re-depicted here in Fig. 1, this work demonstrated thatthe developed characteristics of somatic cells are not fixed(Ladewig et al., 2013; Waddington, 1957). That is, unlike Weis-mann's barrier theory, which stated that unwarranted genetic in-formation is erased in cells committed to a certain state(Weismann, 1893), Gurdon's nuclear transfer experiments showedthat not only do somatic cells maintain all genetic material, butthey can also be reignited by artificial manipulations allowing forthe return to a state of pluripotency. Shortly after Gurdon'sgroundbreaking work with nuclear transfer, many other groupsstarted to present more novel findings pertaining to stem cells. Dr.James Till and Dr. Ernest McCulloch provided data to prove theexistence of stem cells by injecting healthy marrow cells into irra-diated mice and observing the presence of undifferentiated cellularcolonies on the recipients' spleens (Till and McCulloch, 1961). Twodecades after Till and McCulloch's finding, Drs. Martin Evans andMatthew Kaufman reported their ability to successfully culturepluripotent cell lines from mouse blastocysts and differentiatethese cells both in vitro and in vivo (Evans and Kaufman, 1981). Inthe following decade, Dr. Ian Wilmut made international headlines

when he used Dr. Gurdon's nuclear transfer method to clone Dollythe sheep by replacing the nucleus of a fertilized embryo with thenucleus of an adult mammary gland cell (Campbell et al., 1996). In1998 Dr. James Thomson further contributed to the stem cell fieldby successfully isolating human embryonic stem cells (Thomsonet al., 1998). Together, these findings along with many other re-ports paved the way for modern stem cell research and regenera-tive medicine.

1.1. Stem cells

Stem cells are defined as cells that have an unlimited capacityfor self-renewal and are capable of differentiating into multiple celltypes. They are often categorized according to 1) potency, or the

L.A. Wiley et al. / Progress in Retinal and Eye Research 44 (2015) 15e35 17

degree or capacity in which they can differentiate into multiplecells types or lineages, and 2) origin of isolation.

1.1.1. Cellular potencyTotipotent 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 tissueand thus the embryonic cellular lineages: ectoderm, mesoderm,and endoderm. Multipotent stem cells have the ability to differ-entiate into a limited number of cell types, which is controlled by apredetermined level of earlier differentiation.

1.1.2. Origin of isolationStem cells may also be defined based on their origin. Human

embryonic stem cells (ESCs) are harvested from the inner cell massof the blastocyst of 5-day-old pre-implantation embryos. As ESCsare pluripotent, i.e. can differentiate into the three germ layers, theyare a popular tool used in regenerative medicine. Fetal stem cells,unlike ESCs, are isolated later in development, i.e. collected fromfetal 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, aresuggested to be present in small numbers in most major organs.These cells can be multipotent, i.e. give rise to a limited number ofcell types specific to the tissue from which they were derived. Forexample, ASCs can be found in bone marrow, intestinal crypts andin the eye near the limbal region posterior to the cornea (Davangerand Evensen, 1971; Jiang et al., 2002; Snippert et al., 2010).

ESCs have played a substantial role in disease modeling andtreatment studies in the past decade. However, harvesting ESCsfrom the inner cell mass of the blastocyst during development re-stricts their clinical practicality due to limited availability andethical concerns. The same can be said for fetal cells, which aretypically isolated even later in embryonic development. In addition,as ESCs and fetal cells are by definition non-autologous, i.e. notderived from the patient for which they are destined, for celltransplantation an additional obstacle of immune incompatibilityexists. For strategies focused on the use of these cell types, life long

Fig. 2. Reprogramming somatic cells into iPSCs. The Yamanaka reprogramming factors (Klf(pink cells). The reprogramming factors may be packaged in lentiviral vectors, which integraThe transduced cells are reseeded onto feeder cells, such as mouse embryonic fibroblastsleukemia inhibitory factor (LIF). Colony formation (green cells) will occur approximately onand subsequently expanded into iPSC lines.

immune suppression may be required to prevent immune-mediated rejection.

1.2. Induced pluripotent stem cells (iPSCs)

Shinya Yamanaka and colleagues revolutionized the stem cellfield in 2006 when they demonstrated that murine fibroblastscould be reprogrammed into ESC-like pluripotent stem cells,termed induced pluripotent stem cells (iPSCs) (Yamanaka andTakahashi, 2006). They used the knowledge that somatic cellreprogramming can be achieved by transferring a somatic cell'snucleus into an oocyte in order to investigate if the factors neededto maintain ESC identity could perform vital functions in theinitiation of pluripotency in somatic cells. Many transcription fac-tors, including Oct3/4, Sox2, and Nanog, work to maintain plurip-otency in early embryos (Avilion et al., 2003; Chambers et al., 2003;Mitsui et al., 2003; Nichols et al., 1998). Other genes that arecommonly up-regulated in tumors, like Stat3, E-Ras, C-myc, Klf4,and b-catenin, are also known to be involved in the long-termmaintenance of ESC pluripotency and the rapid proliferation ofESCs in vitro (Cartwright et al., 2005; Kielman et al., 2002; Matsudaet al., 1999; Phillips et al., 2012). Yamanaka successfully identifiedfour transcription factors, Klf4, Oct3/4, Sox2, and c-Myc (KOSM),which were sufficient for successful reprogramming of mouseembryonic and adult fibroblasts back to a pluripotent state (Fig. 2).Shortly after the original publication, Yamanaka demonstrated thatthis process could be repeated using human fibroblasts, thusmaking his work relevant to human disease and an incrediblypromising potential resource for cellular transplantation studies(Takahashi et al., 2007). Following these original publications, aplethora 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 aimedat reducing the need for virally induced genetic insertion of thereprogramming factors, especially the potentially tumorigeniconcogene, c-Myc.

4, Oct4, Sox2, c-Myc) are virally transduced into somatic cells, like murine fibroblastste into the host DNA, or in Sendai viral vectors, which do not require DNA integration.(MEFs; blue cells), and fed iPSC induction media containing the pluripotency factor,e week post-transduction, allowing for the individual colonies to be manually isolated


The discovery of the iPSC ushered in a new age in the field ofregenerativemedicine. Unlike ESCs, the human iPSC is not hinderedby ethical disputes, and, although not yet fully tested, they shouldbe safer immunologically. That is, a key advantage of the iPSCtechnology is that these cells can be generated in large numbersusing cells taken from the patients for which they are intended.Hence, iPSCs are patient-specific. Additionally, like ESCs, iPSCs arepluripotent and can be differentiated into any cell type of the threeembryonic germ layers.

The ability to generate patient-specific iPSCs creates novelavenues for disease modeling and transplantation studies.Although the iPSC technology holds great promise, a concern ofthis technology is that the transgenes used for reprogrammingcould remain constitutively active or be randomly incorporatedinto the host genome. Retro- and lentiviral platforms commonlyused to deliver reprogramming factors to somatic cells requiresDNA to be integrated into the host's genome. Such genomicintegration can disturb the normal expression and function ofhost genes. These concerns have, in large part, been addressedby use of non-integrating delivery systems such as episomalviruses (e.g. Sendai virus), which allow reprogramming factors tobe introduced into somatic cells without integrating into thehost genome (Ban et al., 2011; Fusaki et al., 2009; Tucker et al.,2013a). Unlike MMLV-based retroviruses or HIV-based lentivi-ruses, Sendai viruses are RNA-based and function independentlyof host cell DNA replication. Consequently, transduction withSendai virus is transient and viral particles can be removed fromthe infected cells following passage and sub-cloning (Ban et al.,2011; Fusaki et al., 2009). In addition to non-integrating viraldelivery systems, several non-viral reprogramming protocolshave also been developed. The most promising of these aretransient transfection with episomal plasmid DNA (Fontes et al.,2013; Hu and Slukvin, 2013; Okita et al., 2013) and synthetic self-replicating RNA (Yoshioka et al., 2013). The latter, developed bymodifying a noninfectious self-replicating Venezuelan equineencephalitis viral RNA replicon, is a stable DNA-independentsingle-stranded RNA molecule that was engineered to carry 4

Table 1Major stem cell categories and their advantages and disadvantages for development of o

Category

Embryonic/fetal Adult

Derivation/generation Derived from developing embryos(e.g. blastocyst or developing fetus)

Derived from d

Examples � Embryonic stem cells� Retinal progenitors� Subventricular zone stem cells� Photoreceptor precursor cells

� Mesenchyma� Hematopoiet� Intestinal cry� Skin derived� Limbal stem

Advantages � Able to generate retinal cells,including photoreceptors, RPE,and choroidal endothelial cells

� Have unlimited self-renewal,so single donations offer enoughcells for large number of transplantations

� No ethical co� Potential to b

for immune� Do not form

Disadvantages � Ethical concerns regarding source� Not patient-specific� Have the potential to form tumors if not

properly differentiated and isolated

� Limited pote� Limited capa

separate reprogramming factors (Yoshioka et al., 2013).Following a single transfection of human fibroblasts efficientgeneration of integration free iPSCs was achieved (Yoshiokaet al., 2013).

To give a concise overview of the 3major categories of stem cellsand their advantages and disadvantages from an ophthalmologicalstandpoint, a summary of the above-described points is presentedin Table 1.

2. Disease modeling using iPSCs

2.1. Mendelian retinal diseases

Being able to offer clinical intervention for inherited geneticdisorders is greatly dependent on the understanding of a patient'sdisease-causing mutations and the consequential pathophysiolog-ical mechanism(s) that these disease-causing mutations induce.Lacking the knowledge of a patient's disease-causing gene and howthe genetic mutations impact the health and behavior of theaffected cells, it would be nearly impossible to generate and pro-vide patient-specific therapeutic strategies. Apart from being apromising cell source for autologous transplantation, a majoradvantage of the iPSC technology is that it provides the ability tomodel and study human disease in vitro. In particular, iPSCs are auseful tool for 1) discovery and molecular confirmation of newly-identified disease-causing mutations; 2) the interrogation of dis-ease pathophysiology in relatively inaccessible tissues such as theretina that cannot be routinely subjected to molecular analysis inliving patients; and 3) the rapid testing of novel disease andpatient-specific therapeutics, especially important for rare diseasesin which animal models that accurately recapitulate the diseasephenotype do not exist.

As depicted in Fig. 3, the ability to develop a better under-standing of disease mechanism will increase the number of ther-apeutic avenues available to clinicians. The following sectionsprovide some examples of the ways in which iPSCs have been usedto model Mendelian eye diseases such as MAK-associated retinitis

cular therapy.

Induced

evelopmentally mature organs Generated from terminally differentiated tissue

l stem cellic stem cellpt stem cellprecursor cellcells

� Induced pluripotent stem cell

ncernse patient-specific, less concernrejectiontumors

� Capable of generating autologouspatient-specific retinal cells includingphotoreceptors, RPE, and choroidalendothelial 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 oftherapeutic efficacy

ncycity for self-renewal

� Have the potential to form tumors if notproperly differentiated and isolated

� Depending on disease phenotype,genetic correction of patient specificmutations may be required

Fig. 3. Schematic depicting how therapeutic modalities available depend on knowl-edge and progression of retinal degenerative disease in question. A major goal of usingpatient-specific iPSCs for disease modeling is to acquire more knowledge of the dis-ease's pathogenesis to effectively prevent the retinal degeneration from progressing.The greater the amount of knowledge known about a particular retinal degenerationallows for the development of preventative therapies. For instance, if a disease-causingmutation is known, pre-implantation testing prior to in vitro fertilization could beperformed to implant ESCs without the disease-causing mutation. However, correctivetreatments are needed as the amount of disease knowledge decreases (green arrow)and disease progression occurs (purple arrow). These avenues start with drug thera-pies that may slow disease progression. If the genetic cause of a disease is known, geneand/or cell replacement therapy may be a viable option. For late-stage disease, moreinvasive therapies like optogenetics or retinal prosthesis may be necessary.


pigmentosa, Usher Syndrome Type II, CEP290-associated Lebercongenital amaurosis, juvenile neuronal ceroid lipofuscinosis, andBest disease, as well as the complex heterogenous disorder, age-related macular degeneration.

2.2. MAK-associated retinitis pigmentosa

Although some inherited retinal diseases are well-understoodmechanistically, like ABCA4-associated Stargardt disease, there arestill a large number of patients whose disease-causing genes areeither unknown or who have known mutations in genes withpoorly understood functions. Retinitis pigmentosa (RP) is a partic-ularly devastating type of blinding disease because it graduallyclaims a person's vision over many years and in some cases alsothreatens the vision of family members. One of the many obstaclesto making gene- and cell-based therapies an everyday reality forpeople afflicted with RP is the extensive genetic heterogeneity ofthe disease. To date there are over 60 different genes that have beenassociated with RP, which can occur in the retina alone or togetherwith other syndromic disorders (Petrs-Silva and Linden, 2014). Inour experience, these 60 genes account for less than half of all casesof the disease. That is, over 163 different genes that cause an RP-likephenotype have been identified via genetic sequencing of patientsat the University of Iowa Carver Non-profit Genetic Testing Lab(unpublished data). Thus, it is not uncommon for a newly discov-ered RP gene to cause less than 1% of all cases of the disorder. Tomake a major impact in the RP field, scientists will need to developways to rapidly translate a success achieved in treating one geneticsubtype of RP into the treatment of another. That is, theywill need aseries of interchangeable and reusable reagents and strategies.Recently we used patient-specific iPSC-derived photoreceptorprecursor cells generated from patients with retinal degenerativediseases to show that a homozygous Alu insertion, identified inexon 9 of the gene male germ cell-associated kinase (MAK), resultsin loss of the transcript and an inability to produce functional MAKprotein (Tucker et al., 2011b). Loss of MAK was found to disruptnormal photoreceptor cell structure leading to cell death andirreversible blindness. In addition to allowing us to confirm path-ogenicity of the MAK mutation, we were also able to identify andconfirm the involvement of a uniqueMAK-specific retinal transcriptin disease pathophysiology. This transcript contains an extra 75base pair exon (exon 12), which is phylogenetically conserved andis only expressed in transcripts that also contain MAK exon 9(Tucker et al., 2011b). Although mutations in MAK are an uncom-mon cause of RP in the general population (about 1%) they are quitecommon among individuals of Ashkenazi Jewish ancestry, ac-counting for as much as one third of this disease in the Jewishpopulation (Stone et al., 2011). Collectively these findings haveenabled progress in the development of gene and autologous cell-based interventions for MAK-associated RP in which patient-specific iPSC-derived photoreceptor cells can be used to confirmtherapeutic efficacy in vitro and replace lost cells in vivo. As anadditional example of iPSC-derived retinal cells being used to testgene-based therapeutics, a recent study published by Tsang andcolleagues show efficacy of AAV8-mediated delivery of MembraneFrizzle-related Protein (MFRP) to rescue the cellular phenotypeobserved 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 cellswere used to confirm two disease-causing mutations, one being anovel intronic mutation in the gene USH2A in an adult patient withautosomal recessive RP (Tucker et al., 2013b). In this study,keratinocyte-derived iPSCs were differentiated into multi-layereyecup-like structures that contained an outer layer of RPE andan inner layer of photoreceptor-like neuronal cells. These photo-receptor precursor cells expressed photoreceptor-specific markers,recoverin and rhodopsin and exhibited axonemes and basal bodiesstructurally characteristic of photoreceptor outer segments (Tucker


et al., 2013b). Further analysis of the identified USH2A transcripts inthese cells revealed that one of the patient's mutations causesexonification of intron 40, a translation frameshift and a prematurestop codon. Increased expression of the endoplasmic reticulum(ER) stress-related proteins, GRP78 and GRP94 suggested that thepatient's other USH2A variant (a single point mutation resulting inan Arginine to Histidine transition at position 4192) causes diseasethrough protein misfolding and ER stress. To further support theidea that this disease is ER stress-related rather than a develop-mental defect, photoreceptor precursor cells were transplantedinto 4-day-old immunodeficient Crb1�/� mice. Transplantedpatient-derived cells integrated and formed morphologically andimmunohistochemically recognizable photoreceptor cells, sug-gesting that the mutations in this patient cause degeneration ofdevelopmentally normal photoreceptors over time. The role of ERstress in RP-associated photoreceptor cell death is consistent withprevious findings in other disease causing mutations. For example,in a similar study Jin and colleagues were able to show that iPSC-derived rod photoreceptor precursor cells generated from a pa-tient with autosomal dominant RHODOPSIN-associated RP haveelevated levels of ER stress-related proteins and subsequentlysuccumb to apoptotic cell death in vitro (Jin et al., 2011). UnlikeMAK-associated RP, neither USH2A nor RHODOPSIN-associateddiseases will be amenable to the traditional gene augmentation-based approach (Burnight et al., 2014; MacLaren et al., 2014;Maguire et al., 2009, 2008; Vasireddy et al., 2013). That is, with acoding region of ~19,000 bp, full-length USH2A exceeds the pack-aging capacity of either of the two viral delivery systems used in theeye to date (i.e. AAV packaging limit is ~4000 bp, lentiviral pack-aging limit is ~10,000 bp). For this disease, delivery systems withlarger carrying capacity could be effective. That said, cloning ofmutation-free genes of this size is often problematic. For autosomaldominant diseases such as RHODOPSIN-associated RP, in which thedisease-causing mutation causes a gain-of-function, deletion orknockdown of the mutated allele will likely be required. As dis-cussed in detail below, a promising new approach that may beuseful for treating both of these classes of disease is CRISPR-basedgenome editing.

2.4. CEP290-associated Leber congenital amaurosis

Leber congenital amaurosis (LCA) refers to a collection ofinherited retinal degenerative disorders characterized bynystagmus, little to no recordable electroretinogram, and poorvision presenting from birth (Coppieters et al., 2010). LCA is pre-dominantly inherited in an autosomal recessive manner and of theseventeen loci associated with the disease, the largest contributoris CEP290 (Perrault et al., 2007; Stone, 2007). Thirty percent of LCApatients carry mutations in CEP290, the gene product of which is acilia-associated protein that functions in photoreceptor outersegment trafficking and ciliogenesis (Craige et al., 2010; Drivaset al., 2013; McEwen et al., 2007; Tsang et al., 2008). Loss ofphotoreceptor outer segments in patients with CEP290-associatedLCA results in severe vision loss. However, some patients retaincone photoreceptors in the central fovea and MRI studies indicatedthat visual pathways of the inner retina in these patients displaynormal architecture (Cideciyan et al., 2007). These data, as well asthe recent successes garnered in gene augmentation therapy trialsfor RPE65-associated LCA, support gene and cell therapy as an op-tion to treat CEP290-associated LCA. Patient-specific iPSC-derivedphotoreceptors provide an excellent opportunity to study thera-peutic strategies in vitro.

Recently, we demonstrated a ciliogenesis defect in CEP290-associated LCA patient cells (Burnight et al., 2014). Previous workindicated that the rd16 mouse model of CEP290-associated LCA

exhibited defects in number and length of cilia in serum-starvedfibroblasts (Drivas et al., 2013). When we investigated ciliogenesisin CEP290-associated LCA patient fibroblasts with various CEP290mutations, we observed a decrease in the number of cells formingcilia and in the length of cilia formed. This cilia phenotype was notobserved in cells from every CEP290 patient, presumably due to thelocation and severity of the mutation within the gene. In addition,following gene augmentation, we were able to show that the rangeof therapeutic dosage was very narrow, i.e. overexpression of wild-type CEP290 itself was toxic. The ability to determine pathophysi-ology and mutation-specific disease severity in individual patientswill give us the opportunity to tailor treatments thereby providingmore efficacious outcomes.

2.5. Juvenile neuronal ceroid lipofuscinosis

In addition to gene and new gene mutation discovery, iPSCs canbe utilized to interrogate disease mechanism, discover potentialdrug therapies and to evaluate the efficacy of gene correction ap-proaches in human cells. We are currently using iPSCs to gaininsight into the devastating autosomal recessive inherited disease,juvenile neuronal ceroid lipofuscinosis, more commonly known asBatten disease. Batten disease is a rare and fatal lysosomal storagedisorder that is most often caused by a one-kilobase genomic DNAdeletion in the gene, ceroid lipofuscinosis 3 (CLN3) (de los Reyeset al., 2004). Ophthalmologists typically diagnose Batten diseaseas it presents with severe deficits in central visual acuity due to lossof cone photoreceptors in the macula. Batten disease is usuallydiagnosed early in a child's life, between the ages of five to seven.Although visual defects and blindness are the first to arise, patientseventually experience seizures, difficulty communicating andwalking and severe mental deficit due to extensive central nervoussystem neuronal cell death. Batten patients face an extremelydifficult life of blindness, epilepsy, are often bedridden and usuallydie during the second or third decade of life. There are currently notreatment options available and little is understood about themolecular role of CLN3 or the mechanism of disease onset whenCLN3 is mutated (Seehafer et al., 2011).

A key feature of Batten disease is the cytoplasmic accumulationof lysosomal autofluorescent material within neurons (lipofuscin)(Gardiner et al., 1990). We are taking advantage of this easilyidentifiable phenotypic disease characteristic to discover drugtreatment options for Batten patients. We have generated patient-derived iPSCs and retinal precursor cells from skin biopsies of hu-man patients with molecularly confirmed CLN3-associated Battendisease. Into these patient-derived cells we are also introducing anapoptosis-dependent reporter construct (Bardet et al., 2008). Weare using these diseased patient reporter cells to perform high-throughput drug screens to look for compounds that are capableof mitigating the accumulation of lysosomal lipofuscin deposits andthat prevent neuronal apoptosis, thus protecting patient-derivedphotoreceptor precursor cells from CLN3-associated cell death.Promising compounds can then be further characterized in vivo fortheir ability to ameliorate lysosomal storage deposits and retinaldegeneration in Cln3 knockout mice (Katz et al., 1999; Mitchisonet al., 1999). Utilizing autologous stem cells for approachessimilar to this one could be a good avenue inwhich to discover newtreatment modalities in addition to providing novel insights intodisease mechanisms for previously untreatable diseases.

2.6. Best disease

Unlike RP, which is usually caused by mutations in genesexpressed in photoreceptor cells, Best disease results from mu-tations in the RPE-specific gene, BEST1 (Petrukhin et al., 1998).


Best disease selectively affects the region of the human retinawithin one disk diameter of the cone photoreceptor-rich foveaand predisposes affected individuals to the development of visionthreatening choroidal neovascular membranes secondary tovitelliform lesions (Kay et al., 2012). Late stages of Best disease areoften marked by geographic loss of the RPE underlying the maculaand gliotic scarring in the neural retina (Mullins et al., 2007,2005). Although mutations in BEST1 have long been known tocause Best disease, as mouse models of disease lack the majordisease phenotypes, the mechanism by which these mutationscause onset of disease remained unclear. In an elegant studyrecently published by Singh et al., human iPSC-derived RPE weregenerated from patients with Best disease and unaffected siblingsand used to examine the cellular and molecular disease processes(Singh et al., 2013). They observed that RPE derived from patientswith Best disease displayed disrupted fluid flux and increasedaccumulation of autofluorescent material after long-term feedingof photoreceptor outer segments (Singh et al., 2013). Furthermore,chronic photoreceptor outer segment feeding led to delayedrhodopsin degradation and turnover, cellular responses to calciumwere sensitized, and oxidative stress levels were altered(Abramoff et al., 2013; Singh et al., 2013), cell biological findingsconsistent with altered outer segment-RPE interactions observedin living Best disease patients (Abramoff et al., 2013). Collectively,these findings suggest that BEST1 plays an important role inphotoreceptor outer segment turnover and homeostasis and im-plicates disruption of this pathway in the pathogenesis of Bestdisease. These studies point to a variety of therapeutic avenues, inparticular drug-based studies focused on manipulation ofphagocytic and protein degradation pathways may be particularlypromising.

2.7. Using iPSCs to model AMD

Age-related macular degeneration (AMD) is a genetically het-erogeneous and complex disease with over 50 different genetic riskloci identified to date (Gorin, 2012; Liu et al., 2012), 19 of whichmeet genome wide significance (Fritsche et al., 2013). Early AMDtypically presents in the sixth decade of life or later with the for-mation of lipid-like deposits, called drusen, that accumulatebeneath the RPE, as well as drusen-like deposits that appear in thesubretinal space (Curcio et al., 2013). A subset of patients with earlyAMD later develops geographic atrophy of the RPE and underlyingchoriocapillaris, resulting in the loss of the overlying photorecep-tors. Other patients with early AMD develop a complicationsometimes referred to as “wet AMD” in which the underlyingchoroidal vasculature grows into the sub-RPE and/or sub-retinalspace forming fenestrated, leaky vascular networks known aschoroidal neovascular membranes (CNVs) (Jager et al., 2008). ForAMD, all of the therapeutic interventions shown in Fig. 3 couldeventually come into play. Drug therapy using the anti-vascularendothelial growth factor (VEGF) agents Avastin and Lucentishave proven very efficacious in ameliorating CNVs, promoting fluidresorption, and slowing vision loss in wet AMD (Agosta et al., 2012;Scott and Bressler, 2013; Stone, 2006). As AMD is geneticallyheterogenous, traditional gene augmentation approaches seem lesspromising than for Mendelian diseases. However, cell replacementtherapy for patients that have lost photoreceptors, RPE andchoroidal (choriocapillaris) endothelial cells is being pursued by anumber of groups and, if successful, would be extremely beneficialfor thousands of patients worldwide (Kamao et al., 2014; Kokkinakiet al., 2011; Liao et al., 2010; Zhu et al., 2011). In very late stage AMD,in which significant cell loss has occurred, optogenetic and retinalprosthetic approaches may eventually be viable therapeutic op-tions, although the resolution of the latter devices will need to be

improved if they are to offer AMD patients a significant improve-ment in visual acuity.

The greatest opportunity for AMD treatment lies in under-standing the genetic causes and pathogenic mechanisms thor-oughly enough to be able to identify those with a greaterpropenstity to develop the disease and prevent it from happeningthrough early and efficacious prophylactic intervention. Althoughthe notion that AMD has a strong genetic component was advo-cated as early as the 1970s (Davanger and Evensen, 1971; Gass,1973; Jiang et al., 2002; Snippert et al., 2010), our understandingof the biological mechanisms through which specific genetic locicontribute to the development and progression of AMD is stillpoorly understood. Genome wide association studies of AMDidentified two loci on chromosomes 1 and 10 that are highly-associated 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 chromosome1q locus is predominantly due to a haplotype that harbors amissense 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 ofAMD prior to the association of CFH variants with the disease (Boraet 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 in-hibitor in AMD fits into a mechanistic framework of impairedcomplement regulation and bystander cell injury (Mullins et al.,2011a; Tucker et al., 2011b). In contrast, the mechanism by whichthe chromosome 10q locus increases AMD risk remains unknownand controversial. The most common high-risk haplotype containstwo genes (ARMS2 and HTRA1) with plausible disease-causingmutations. The variants include a non-conservative poly-morphism in the ARMS2 gene (Ala69Ser), a complex 144 bp dele-tion and a 54 bp insertion in the 30 untranslated region of theARMS2 transcript, and a promoter polymorphism in theHTRA1 gene(Fritsche et al., 2008; Stone et al., 2011).

These variants are in strong linkage disequilibrium such thatonly a few percent of 10q alleles harboring ARMS2 Ala69Ser lack theHTRA1 promoter variant and vice versa. This, coupled with theoverall genetic complexity of AMD and a lack of representativeanimal models, have made it difficult to determine which, if either,of these two genes is responsible for conferring the AMD riskassociated with the 10q locus. Recently, Yang and colleaguesemployed an unbiased proteome screen of N-retinylidene-N-ret-inyl-ethanolamine (A2E)-aged patient-specific iPSC-derived RPEcell lines derived from patients with high and low risk 10q26 loci(Coppieters et al., 2010; Yang et al., 2014). They reported that cellsfrom patients who were homozygous for the high-risk haplotypehad a reduction in superoxide dismutase 2 (SOD2)-mediated anti-oxidative defense. The authors concluded that the ARMS2/HTRA1risk alleles decrease SOD2 defense, leaving RPE more susceptible tooxidative damage and thereby contributing to AMD pathogenesis.By elucidating the disease mechanism(s) associated with this locus,new targets for therapeutic intervention have potentially beenidentified.

Historically, the RPE has been seen as the key cell type involvedin AMD pathogenesis. Currently, clinical trials focused on stem cell-derived RPE cell transplantation are underway. The first, initiatedby Robert Lanza and colleagues at Advanced Cell Technology in theUK, was designed to treat patients with dry AMD and Stargardtdisease. In this trial patients receive bolus subretinal injections ofhuman embryonic stem cell-derived RPE (NCT01344993). Initialshort term reports indicate that transplanted cells attached and

Fig. 4. Choriocapillaris dropout and membrane attack complex in eyes with maculardegeneration. (A) Immunofluorescent labeling of the complement membrane attackcomplex (green) in a human eye with early/dry AMD. Note the robust labeling at thelevel of the choriocapillaris (CC) and relative sparing of the RPE. Sections were duallabeled with UEA-I, a marker of viable endothelial cells (red) in the choroid of an eyewith dry AMD. Note the abundant “ghost” vessels in the choriocapillaris (asterisks),that are unoccupied by viable endothelial cells. Choriocapillaris loss in eyes withadvanced AMD is striking, as depicted in an eye with choroidal neovascularization (B).A comprehensive cell replacement strategy to treat AMD will most likely requireaddressing the vascular loss in this disease. BlamD: layer of basal laminar deposit, RPE:retinal pigment epithelium, CC: choriocapillaris, CH: outer choroid; RET: neural retina.


persist within the subretinal space (Schwartz et al., 2012a). PeteCoffey, Dennis Klegg and colleagues have devised a similar trialfocused on subretinal transplantation of human ESC-derived RPEcell sheets for the treatment of patients with AMD (NCT01691261).Similarly, in Japan, Masayo Takahashi and collaborators at theRIKEN Center for Developmental Biology have recently trans-planted autologous iPSC-derived RPE cell sheets into awomanwithAMD (JPRN-UMIN000011929). The latter two studies have focusedon sheet transplants rather than bolus cell injection, which havebeen shown to dramatically improve survivability and integrativecapacity (Hu et al., 2012). Cell suspension injections may lead to theformation of isolated cellular clumps that fail to develop intopolarized RPE monolayer (Lu et al., 2001; Phillips et al., 2003;Shiragami et al., 1998).

To date, the contribution of the choroid, in particular the un-derlying choriocapillaris vasculature that supports the RPE andphotoreceptor cells, has not been thoroughly studied and remainsunknown. This lack of knowledge is largely due to the historicalinability to clinically image this tissue for better understanding itsrole in pathogenesis. Recent advances in imaging technology nowallow for live imaging, measurement and assessment of choroidalthickness using spectral-domain optical coherence tomography(SD-OCT). Previous studies using the SD-OCT technique on eyeswith early-stage AMD without CNVs showed a thinning of thechoroid 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 inAMD pathogenesis, findings from our group and others have shownthat there is loss of choriocapillaris endothelial cells in eyes withboth early- and late-stage AMD (Grunwald et al., 2005; McLeodet al., 2009; Ramrattan et al., 1994). For instance, Sohn and col-leagues recently reported that human donor eyes known to havedry AMD are associated with choroidal thinning and that AMDchoroids have higher levels of tissue inhibitor of metalloproteinase3 (TIMP3), which is associated with Sorby's fundus dystrophy, anautosomal-dominant form of macular degeneration (Sohn et al.,2014; Visse and Nagase, 2003). Additionally, a gene set enrich-ment analysis (GSEA) study on RPE-specific and endothelial cell-associated gene sets found that RPE transcripts were preservedand increased in early AMD, along with a significant decrease ofendothelial cell marker expression, suggesting that choroidalendothelial cell dropout is an early event in the pathogenesis ofAMD (Whitmore et al., 2013), consistent with morphometricstudies showing increased numbers of choriocapillaris ghost ves-sels with increasing abundance of drusen (Fig. 4A) (Mullins et al.,2011b). These findings indicate that choroidal endothelial cellpreservation may slow the progression of or help protect againstthe onset of early AMD.

Stem cell-derived choroidal endothelial cells for the purpose ofcellular transplantation may also be required for the reconstructionof the macula in advanced AMD. Although the loss of chorioca-pillaris EC in early AMD is notable, in endstage AMD (geographicatrophy or choroidal neovascularization) choriocapillaris dropout isprofound (Fig. 4B). In advanced AMD, a combined approach inwhich photoreceptor cells, RPE and choriocapillaris EC are replacedmay be necessary in order to restore vision.

Endothelial cells can be generated from iPSCs through co-culture with primary endothelial cell lines or conditioned mediawith endothelial cell-specific factors (Choi et al., 2009; Du et al.,2014). For instance, Park et al. successfully generated cord bloodiPSC-derived vascular progenitors and used these cells to repairdamaged retinal blood vessels (Park et al., 2014b). However, to ourknowledge there is no published data showing the differentiationof iPSC-derived choroidal endothelial cells. The choroid is struc-turally distinct from other vascular tissues, like the brain

microvasculature or aortic vasculature, due to its broad, fenestratedand flat lumens with diameters ranging from 20 to 50 mm (Luttyet al., 2010). Nevertheless, the extent to which choroidal endothe-lial cells are molecularly unique compared to other endothelial cellsis not well understood. It is possible that the microenvironment ofthe choroid causes residing hemangioblasts, precursors of he-matopoietic and endothelial cells, to develop and maintain theunique choroidal endothelial cell features. For example, it isthought that VEGF secreted from the overlying RPE is necessary inmaintaining the fenestrations seen in the choriocapillaris


(Blaauwgeers et al., 1999; Lutty et al., 2010). In order to developsuccessful treatments for macular degenerations involvingchoroidal disruption, it is critical to understand if the choroidalendothelial cells vary from other endothelial cells. Therefore, futurestudies are needed to characterize choroidal endothelial cells todetermine if they are unique compared to other endothelial cells atthe molecular level.

Although the replacement of choroidal ECs derived from iPSCsposes many challenges, there is basis to suspect that autologous ECmay be well tolerated in the choroid. IPSC-derived ECs have beenshown to integrate with the host vasculature and trigger a rela-tively modest immune response in a murine model of hindlimbischemia (de Almeida et al., 2014). Transplanted iPSC-derived ECsmay similarly recanalize ghost vessels in the choriocapillaris. This isan active area of investigation.

As mentioned above, complement activation has long beenshown to play a major role in the pathogenesis of AMD. This notionis further substantiated in that the complement cascade is alsoactive in the choriocapillaris of high-risk CFH AMD eyes (Mullinset al., 2011a). It is possible that complement complexes near thechoriocapillaris put stress on the choroidal endothelium and helpcause 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). Theseresults further demonstrate the need to elucidate the choroid's rolein AMD pathogenesis that could contribute to the development ofnew treatments for AMD. Development and implementation ofefficient choroidal endothelial cell differentiation protocols willempower further studies focused on modeling aspects of AMDspecific to the choroid.

Stem cell-derived therapeutic modalities are also showingpromise for the treatment of another blinding eye disease, glau-coma. The laboratory of Donald Zack has used iPSC-derived gan-glion cells to perform high-throughput drug screens for agents thatprevent ganglion cell death in glaucomatous cells and for assessingpotential neuroprotective growth factors secreted by iPSC-derivedretinal ganglion cells (Sluch and Zack, 2014). Also, John Fingertand collaborators recently demonstrated the generation of patient-specific iPSC-derived ganglion cells that have a duplication of thegene, TBK1, which causes normal tension glaucoma. In this studypatient-specific iPSC-derived retinal cells were used to show thatTBK1 duplication causes increased activation of the autophagypathway (Tucker et al., 2014).

In the following sections, we will review drug, gene replace-ment, genome editing and cell replacement strategies beingemployed by our group and others to treat inherited orphan retinaldiseases. Included, will be a discussion regarding the debate of eyeimmune privilege and how donor and autologous stem cell trans-plantation approaches could affect the recipient eye and immu-nological homeostasis.

3. Gene augmentation and cell replacement treatmentstrategies

The heritability of Mendelian retinal degenerative diseases,while one of their most frightening features, is also the means bywhich they will be cured. That is, the fact that these diseases arecaused by detectable variations in a gene that is expressed in theretina will make it possible to 1) lessen the risk of recurrence of thediseases in affected families through a combination of genetictesting and genetic counseling, 2) prevent vision loss by earlydetection of the disease coupled with gene replacement therapy orsome other form of treatment (e.g., immune modulation or drugtherapy) and 3) create genetically-corrected and immunologically-

matched photoreceptor precursor cells that can be transplantedinto the retina to restore vision.

For numerous diseases, especially those involving significantneurodegeneration or other types of organ failure, drug interven-tion or gene therapy alone will not suffice. In these conditions,cellular replacement will be required to rescue, refurbish andpreserve tissue function. Recessive diseases, particularly those thatresult in a null phenotype, such as MAK-associated RP and Battendisease, to name two, are often amenable to gene therapy. That is,replacement of the defective mutated gene with the normal tran-script prior to photoreceptor cell death could potentially arrestdisease progression and prevent subsequent loss of vision. A varietyof gene delivery approaches have been developed and used in theeye. These range from non-viral, plasmid transfection approaches,such as cell-penetrating nanoparticles (Read et al., 2010a,b), to viralvector transduction technologies, such as adeno-associated viruses(AAV) (Maguire et al., 2009, 2008; Narfstr€om et al., 2003; Yanget al., 2002) and lentiviruses (Auricchio et al., 2001; Pang et al.,2006).

As genetic testing for rare recessive diseases becomes morewidespread, patients will be definitively diagnosed earlier in thecourse of their disease when viral gene replacement therapy hasthe greatest opportunity to be efficacious, thus sparing the patient'svision. However, for patients with more advanced disease, retinalcell replacement therapy will be necessary. Patient-derived iPSCshave nowmade this avenue a realistic option. As we have discussedabove, patient-derived stem cells harbor disease-causingmutations(Tucker et al., 2013b, 2011b), which make them ideal for diseasemodeling but a bit less convenient for cell-replacement therapy.That is, for many disorders, the patient's disease-causing mutationsmust first be corrected prior to transplantation if they are going tobe able to have any lasting positive effects. This relative disadvan-tage is balanced by the tremendous advantage that patient-derivediPSCs are immunologically identical to the patient and thus havethe greatest chance of evading the recipient's host immune systemwith the smallest amount of immunomodulatory medication.

3.1. iPSCs and gene augmentation

Gene augmentation is an effective strategy to treat inheritedretinal dystrophies, the causes of which are mutations in enzymesor other small genes easily packaged into the well-characterizedand efficient adeno-associated virus (AAV) vectors. Numerousclinical trials involving AAV-mediated RPE65 augmentation haverestored vision to patients suffering from RPE65-associated LCA e

an inherited retinal dystrophy resulting from deficiency of theretinal isomerase, RPE65 (Bainbridge et al., 2008; Hauswirth et al.,2008; Maguire et al., 2009, 2008). Most recently six patients withthe X-linked recessive blinding disease choroideremia were givenAAV vectors expressing the CHM gene that encodes the Rab escortprotein 1 (REP1). Six-months post-injection, increases in visualacuity and retinal sensitivity (measured via dark-adapted micro-perimetry) were consistent with improved rod and cone function inAAV-treated eyes (MacLaren et al., 2014).

We recently demonstrated successful gene transfer to CEP290-associated LCA patient iPSC-derived photoreceptor precursors us-ing lentiviral-mediated gene delivery (Burnight et al., 2014).Because CEP290 cDNA (~8 kb) is too large to package in AAV vectorscommonly used for clinical gene therapy treatments, we packagedthe full-length CEP290 in a lentivirus which can accommodatelarger transgenes (8e10 kb packaging capacity) (Balaggan and Ali,2012). Unfortunately but importantly, we found that over-expression of this large structural protein caused increased celldeath in a dose-dependent manner of CEP290-expressing lentiviralvectors (Burnight et al., 2014). Nonetheless, when using a lower


dose, we were able to rescue ciliogenesis in diseased patient cellsindicating that restoring full-length CEP290 expression may be aneffective treatment option for vision loss in these patients. Due tothe adverse cellular toxicity associated with CEP290 over-expression, future experiments in gene transfer will need toaddress this obstacle. Using cell type-specific promoters whosetranscriptional activity is less than that of the strong, viral cyto-megalovirus promoter used in our study might decrease thetoxicity resulting from CEP290 gene transfer (Burnight et al., 2014).Additionally, one could take advantage of the native CEP290 regu-latory elements through genome editing strategies to correct thepatient-specific mutation, thereby avoiding ectopic CEP290expression altogether.

3.2. iPSCs and genome editing

Recent advances in genome editing approaches, including theuse of zinc finger nucleases (ZFNs) and transcription activator-likeeffector nucleases (TALENs), are especially exciting for the poten-tial boost these technologies could provide to cell replacementtherapy (Christian et al., 2010; Hockemeyer et al., 2011; Miller et al.,2007; Porteus and Baltimore, 2003). Components of the prokary-otic CRISPR (clustered regularly interspaced short palindromic re-peats)/Cas9 adaptive immune system can also be used to efficientlyand specifically edit human genes using pre-designed 20 nucleo-tide small guide RNAs (sgRNA) coupled with a human codon-optimized Cas9 nuclease (Cong et al., 2013; Maeder et al., 2013;Ran et al., 2013a,b). Similarly to ZFN- and TALEN-mediated genecorrection, cells repair the majority of CRISPR/Cas9-induced doublestrand breaks (DSBs) by one of twomechanisms. In the absence of ahomologous repair template, the cell naturally employs non-homologous end joining (NHEJ) leading to the insertion or dele-tion (InDel) of sequence surrounding the DSB (Sun et al., 2012).Repair via NHEJ represents a feasible strategy for intronic muta-tions in areas of poorly conserved sequence. However, to correctloci at which large deletions or insertions occur or where NHEJ-mediated InDels would not be tolerated (i.e. splice sites), thesgRNAs and Cas9 must be co-delivered with an engineered repairtemplate containing unmutated, wild-type sequence. DSB repairoccurs via homologous recombination using the exogenous repairsequence as template. Homology-directed repair (HDR) in diseasedpatient cells harboring mutations would presumably restore wild-type gene and protein expression and function. Thus, followingCRISPR-based correction, stem cell-derived retinal cells to be usedfor human transplantation would no longer harbor disease-causingmutations. This technology has the potential to elevate stem cellreplacement therapy from being a stopgap, temporary solution, tobeing a long-term disease remedy.

As a proof of principle for the utility of combining genome en-gineering with the iPSC technology, researchers in the Jaenisch labcombined engineered ZFNs with iPSC technology to create diseaseand control human iPSC lines to study Parkinson disease (Soldneret al., 2011). Soldner and colleagues designed ZFNs targeting thea-synuclein locus (SNCA), a gene commonly mutated in Parkinson'sdisease (PD). Using four different targeting strategies, the in-vestigators engineered an array of isogenic and control cell linesthat either created two common PD-related mutations (A53T orE46K) in wild-type ESCs or corrected the mutation in PD patient-derived iPSCs.

Soldner et al. initially employed a drug selection-based strategyto introduce the A53T mutation (a single base pair sub-stitutiondG209Adin exon three of SCNA) into ESCs. The donorconstruct carried ~600 bp homology on either side of the wild-typesequence at nucleotide 209 (G209) and a floxed puromycin-resistance cassette just downstream of the mutation in intron

three. Co-electroporation of the ZFN expression plasmids anddonor construct into two ESC lines resulted in 3 out of 336puromycin-resistant clones targeted to the correct locus. FollowingCre-mediated excision of the selection cassette and sequenceanalysis, two of the three clones contained a small deletion in thesecond allele as a result of the modification. However, theremaining correctly targeted clone maintained pluripotency andwas able to differentiate into tyrosine hydroxylase-expressingneurons (Soldner et al., 2011). The second strategy utilized bothpositive and negative selection to introduce the A53Tmutation intoboth alleles of one of the ESC lines. By incorporating the herpessimplex virus thymidine kinase (HSV-TK) and diphtheria toxin A(DT-A) into the donor cassette, 9 of 41 puromycin- and ganciclovir-resistant colonies resulted in correct targeting. One of the clonesconfirmed by Southern DNA and sequencing analysis containedmodification of the second allele.

A third strategy generated A53T mutant ESCs without drugselection. The investigators co-delivered ZFNs and a donorplasmid carrying about 1 kb homologous sequence flanking theZFN cleavage site and the A53T (G209A) point mutation. Addi-tionally, Soldner et al. delivered a reporter cassette expressingeGFP to enrich transfected cells via fluorescence-activated cellsorting (FACS). Analysis of single-cell-derived clones via Southernblotting and PCR-mediated genotyping revealed one of 240clones was correctly targeted. Cells from the clone maintainedpluripotency and were able to differentiate into dopaminergicneurons (Soldner et al., 2011). Instead of using a double-strandedplasmid donor vector, the fourth strategy employed a shortsingle-stranded oligodeoxynuceotide (ssODN) donor construct asa repair template to introduce the PD-associated E46K (G188A)point mutation into ESCs. Using the aforementioned FACSenrichment strategy, the researchers successfully recovered fiveof 720 single-cell-derived clones with the correct insertion of theE46K/G188A mutation.

Lastly, the investigators employed ZFN-mediated genome edit-ing to repair the A53T mutation in PD patient-derived iPSCs. In theabsence of drug selection, Soldner et al. delivered ZFNs targetingthe G209A mutation in exon three of SNCA and a wild-typesequence-containing donor template carrying ~1 kb homologysequence flanking the targeting site. One of 240 single-cell-derivedclones contained the correctly repaired nucleotide at position 209of SCNA. Subsequent pluripotency marker expression analysis andteratoma formation assays indicated that the genetically correctedclonemaintained pluripotency. Moreover, cells from the clonewereable to differentiate into tyrosine hydroxylase-expressing dopa-minergic neurons that no longer expressed the mutant transcript.This elegant set of experiments demonstrates the utility incombining genome editing strategies with iPSC technology tointerrogate disease-causing mutations and therapeutically correctthem in vitro.

TALENs were used to modify the murine coagulation factor VIII(F8) locus in iPSCs (Park et al., 2014a). Mutations in this locus inhumans cause one of the most common bleeding disorders, He-mophilia A. Park et al. engineered TALEN pairs targeted to intron 1of the murine F8 gene and electroporated plasmids encoding themost efficient pair into wild-type iPSCs. After clonal selection andPCR analysis, the researchers recovered six of 432 (1.4%) colonieswith a 140 kbp inversion resulting from non-allelic homologousrecombination (HR), thus creating model cell lines containing acommon mutation in patients with Hemophilia A. Expressionanalysis in the inversion clones indicated loss of F8 mRNA andprotein. Importantly, Park and colleagues were able to restore F8expression through inducing reversion of the 140 kbp segment bydelivering the same TALEN pair to the mutant clones (Park et al.,2014a). Thus, the researchers demonstrated the use of genome


editing technologies to both model and correct genetic diseases inthe culture dish.

Recently, Wu and colleagues utilized the CRISPR-Cas9 systemto correct a dominant mutation in the murine Crygc gene e mu-tations in which cause cataracts (Wu et al., 2013). Co-injection ofCas9 mRNA and an sgRNA targeting the mutant allele with one oftwo oligonucleotide repair templates into Crygc mutant embryosyielded HDR-mediated repair in 5 of 29 (Oligo-1; 17.2%) and 4 of27 (Oligo-2; 14.8%) live-born pups. All mice that carried the cor-rected allele induced by HDR were free of cataracts and the lenseswere histologically normal compared to non-rescued mutant mice(Wu et al., 2013). These results demonstrated that the CRISPR-Cas9system could be used to correct genetic disease phenotypesin vivo.

While the combination of genome editing strategies with iPSCtechnology represents a powerful avenue by which to investigatethe pathophysiology of inherited retinal dystrophies as well as ef-ficacy of treatment, there is concern that DSB induction at off-targetsites will create adverse effects such as genotoxicity. Indeed, theCRISPR/Cas9 system can tolerate up to five mismatches in thesgRNA 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 essentialwhen employing genome editing in the clinic. The majority ofbioinformatically predicted off-target modifications can beassessed through PCR amplification and Surveyor nuclease diges-tion (Fig. 5). Surveyor nuclease is sensitive enough to detect fromabout three percent modifications at a locus (Qiu et al., 2004),however, genome wide analysis using deep sequencing technolo-gies allows for thorough assessment of safety in modified cells.Several groups have performed off-target analyses using thesemethods and results indicate that modification at unintended locioccurs 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 inCRISPR-Cas9 modified mice via DNA sequencing of PCR productsfrom ten potential off-target sites in twelve mice. No mutations

Fig. 5. Off-target mutation analysis in cells treated with CRISPR/Cas. A) Schematicrepresentation of the EMX1 locus. A previously described sgRNA targeting just up-stream of exon 3 (red arrow) of EMX1 (sg5) (Ran et al., 2013a) was delivered with thewild-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 (Conget al., 2013). The top sites are shown in the table. Bolded letters indicate basechanges from the sgRNA, sg5. C) Surveyor nuclease activity at the on-target (sg5) andfour off-target (OT) sites in duplicate. Genomic DNA from cells treated with sg5 (þ) oruntreated controls (�) were subjected to Surveyor nuclease detection analysis (Ranet al., 2013b). sgRNA: small guide RNA, PAM: protospacer adjacent motif, OT: off-target.

were recovered in ten of the twelve mice evaluated. Of theremaining two, only one site carried off-target modifications (Wuet 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 theenzyme to a ‘nickase’, i.e. Cas9 cleaves only the strand noncom-plementary to the guide RNA. Single-stranded nicks are repaired bythe base excision repair pathway (BER) (Dianov and Hübscher,2013), and thus maintain genome integrity. By delivering theCas9-nickase guided by a pair of sgRNAs targeting opposite strandsof the target locus, the nicking enzyme creates DSBs with minimaloff-target modification. Ran and colleagues recently demonstrateda 50e1500-fold reduction in off-target cleavage using thisapproach. Importantly, employing the nickase strategy maintainson-target activity (Ran et al., 2013a). Similarly, shortening thesgRNAs can reduce off-target activity. Fu et al. showed that sgRNAsof 17 or 19 nucleotides in length reduced off-target activity by asmuch as 5000-fold in some cases (Fu et al., 2014). Again, thesetruncated guides showed comparable on-target activity. Employingeither truncated guides or paired nickases, or both strategies inconcert can greatly reduce undesired off-target effects. Theseexciting technologies, in addition to future developments ingenome editing, will be essential in allowing immunologically-matched and genetically wild-type cell replacement in heritableforms of blindness.

3.3. iPSCs for testing of therapeutic efficacy

Although human stem cells represent an exciting new frontierfor treatment of inherited blinding diseases, a sobering aspect ofthis otherwise exciting progress is the sluggish pace at which thefew therapies that are available today have moved from “proof-of-concept” stages in animals to a fully-approved treatment availableto patients who need them.

An example of this is the substantial amount of time it took forRPE65 gene replacement therapy to become a reality for patientsafflicted with RPE65-associated LCA. Efficacy of RPE65 genereplacement was demonstrated in a canine model in 2001 (Aclandet al., 2001), and over thirteen years later, fewer than 100 patientshave been treated world-wide and the Phase III clinical trial is stillongoing. This raises the possibility that some diseases might be sorare in the population that it is not economically logical and feasibleto bring a treatment through the regulatory gauntlet and intoclinical availability. One possible solution for disorders that are sorare that they are below the commercial viability economicthreshold is to use autologous patient-derived cells to test themolecular efficacy of a drug or viral-mediated gene therapy.Although the optimal dosages of therapies may not be identical inculture and in vivo, demonstrating efficacy and a range of toleratedand effective doses in patient-specific cells will be an importantstep forward. We envision that the identified therapies can then bedelivered to one eye in a compassionate use manner. Patientstreated in this way can be monitored at intervals using conven-tional clinical measures such as visual acuity, Goldmann perimetry,ganzfeld electroretinography and optical coherence tomography. Inthe event treated eyes fare worse than untreated ones, thecompassionate use treatment can be stopped until the reason forthe poorer outcome can be identified and an improved treatmentcan be developed. When treated eyes display noticeable improve-ment in vision than untreated eyes over time, the treatment canthen be offered to new patients and the second eyes of the initialpatients can also receive treatment. In adopting a treatmentapproach like this, a series of increasingly predictable, reusable

http://crispr.mit.edu


parts can be assembled and used to devise a compassionate usetherapy for retinal disorders, regardless of their rarity.

3.4. iPSCs and drug discovery

For translational applications like discovering novel pharma-cotherapeutics, human stem cell-based assays represent a physio-logically relevant and high-throughputmeans to assess efficacy andtoxicity early on in the drug development pipeline. Human stemcells are a means for expediting early proof-of-concept studies forpotential drug candidates. As indicated above, retinal cells derivedfrom iPSCs are faithful representatives of their in vivo counterparts(Tucker et al., 2013b). High-throughput drug screens of differenti-ated iPSC-derived cells have been suggested as a means for drugdiscovery and personalized medicine (Desbordes and Studer, 2013;Inoue and Yamanaka, 2011). Drug screening using extra-oculariPSC-derived cells from patients with rare diseases has shownexcellent promise. Screens have been performed on iPSC-derivedcardiomyocytes (Cerignoli et al., 2012; Mordwinkin et al., 2013),forebrain neurons (Luo et al., 2013; Yahata et al., 2011), motorneurons (Egawa et al., 2012), and hepatocytes (Kim et al., 2013), toname a few examples.

Combining the power of differentiated ocular cells with high-throughput screens will likely prove effective for discovering newpharmacologic treatments for delaying the progression of retinaldegenerative diseases. High-throughput drug screens will need tobe highly organized and well thought out, however, if they are tosuccessfully identify reliable drug treatment candidates. Screeningassays will need to be multiplexed to simultaneously assess theproliferation, morphology, differentiation state, viability and func-tion of specific iPSC-derived retinal cell types that would enable themonitoring and testing of disease-relevant phenotypes (Wrightet al., 2013).

To date there have been a limited number of reports looking atthe effects of drugs on iPSC-derived retinal cells. David Gamm andcollaborators reported the restoration of ornithine aminotrans-ferase activity in iPSC-derived RPE from a patient with gyrate at-rophy following treatment with vitamin B6 (Meyer et al., 2011a). Ithas been known for some time that vitamin B6 is beneficial forpatients with gyrate atrophy. However, the patient of interest inthis study was previously unresponsive to vitamin B6 supplemen-tation. This study highlighted the use of iPSC-derived RPE to testthe specific cellular population targeted in gyrate atrophy to moreaccurately test vitamin B6 efficacy.

In another study, the lab ofMasayoTakahashi generated patient-specific iPSC-derived rod photoreceptor-like cells harboring mu-tations in several genes that cause retinitis pigmentosa like RP1,PRPH2, RHODOPSIN, and RP9 (Jin et al., 2011). This study showedthat diseased RHODOPSIN-positive iPSC-derived retinal cells peri-shed after 120e150 days in the culture dish. The authors thentreated cells with 3 antioxidant vitamins, a-tocopherol, ascorbicacid and b-carotene. They observed that cells treated with a-tocopherol led to a statistically significant increase in survival ofdiseased iPSC-derived photoreceptor lines, providing proof-of-principal for drug screening of iPSC-derived retinal cells.

IPSC technology offers an unlimited source of disease- andpatient-specific material for automated, high-throughput drugscreening systems to screen libraries of drug compounds. As evi-dence for the efficacy of this strategy, work from the laboratory ofDonald Zack, which utilized a high-throughput RNA interferencescreen and primary mouse retinal ganglion cells, demonstratedthat large compound libraries could be effectively screened in arapid fashion to identify novel therapeutics that promote retinalganglion cell survival (Welsbie et al., 2013). Ultimately, replacingprimary neurons in these experiments with iPSC-derived retinal

cells will enhance the efficiency of drug discovery and decrease thetime lag between compound discovery and Phase I clinical trials.

3.5. Retinal transplantation and cellular replacement

Cell replacement therapy is simply defined as replacing dead ordamaged cells with healthy cells to restore the function that aspecific cell population provides within a tissue. The retina is a goodcandidate 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 deliverythrough subretinal injection, a standard technique that can beperformed by most vitreoretinal surgeons. Clinical diagnosis andpost-intervention examining of the retina are also feasiblecompared to other regions of the CNS due to the transparency ofthe cornea, lens, and intraocular media.

However, there is a longstanding argument amongst cliniciansand vision scientists pertaining to the following key questions: 1)What is the optimal stem cell source for production of outer retinalcells?; 2) What is the optimal stage of differentiation for post-transplant survival, integration and cellular function?; and 3)How will the local diseased environment respond to transplantedcells immunologically?

1) What is the optimal stem cell source for production of outerretinal cells?

Retinal progenitor cells (RPCs) have been investigated as a po-tential option for retinal cell replacement therapy. These cells canbe isolated from early embryonic tissue and give rise to ganglion,amacrine, horizontal, retinal pigmented epithelium, and photore-ceptor 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 trans-plantation of GFP-positive RPCs into retinal degenerative mousemodels results in migration of the transplanted cells into the outernuclear layer, differentiation into immunohistochemically identi-fiable rod photoreceptor cells and functional rescue of blindness inthe form of improved pupillary light responses (Klassen et al.,2004). Likewise, transplantation of human RPCs into the sub-retinal 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 andas such are limited in number, rife with ethical concerns and areimmunologically dissimilar from the transplant recipient.

In light of this, the two most promising stem cell types for hu-man retinal transplantation are human embryonic stem cells (ESCs)and induced pluripotent stem cells (iPSCs). As indicated above, ESCsare harvested from developing embryos approximately 4e5 daysafter fertilization just prior to implantation (Thomson et al., 1998),whereas iPSCs can be generated by reprogramming various celltypes with cocktails of transcription factors, such as adult dermalfibroblasts via viral transduction of the transcription factors Oct4,Sox2, Klf4, and c-Myc. Both ESCs and iPSCs can be expanded to yieldsufficient cell numbers for clinical applications and both have thecapacity to generate all of the various cell types of the retina. Todate, differentiation protocols capable of generating photoreceptorprecursor 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 cellpopulation, but the harvesting and use of ESCs is highly contro-versial from an ethical point of view, and like RPCs, ESCs would bean allogenic transplanted cell population, as donor and recipientwould not be immunologically matched. This situation could have


negative consequences within the retina following transplantation,including but not limited to, rejection of the transplanted cells andan unwanted inflammatory response in the eye. On the other hand,iPSCs represent an autologous donor population because they aregenerated from somatic cells isolated from the patient for whichthey are intended (e.g. skin-derived fibroblasts or keratinocytes).However, as we will describe further below, the retinal immuneenvironmentmay bemore complicated, and even this cell typemayrequire post-transplant immunemodulation. A drawback to the useof autologous iPSCs is that since they are generated on a patient-specific basis, they will take much longer to produce and as theywill be produced ‘as needed’ they will require significantly moremanpower and resources to meet clinical demands. Additionally,depending on the patients' original disease-causing mutation, ge-netic correction may also be required. Although timewill tell whichof these cell types will be optimal for therapeutic cell replacement,we have chosen to hedge our bets with the autologous iPSC-basedstrategy.

2) What is the optimal stage of differentiation for post-transplantsurvival, integration and cellular function?

The next big question after deciding which stem cell source toutilize is at what stage of differentiation should cells be in order toachieve optimal levels of post-transplant survival, integration andfunction? For photoreceptor cell transplantation, the post-mitoticphotoreceptor precursor cell has been shown to be at the optimalstage of development for retinal transplantation (i.e. it possessesthe greatest capacity for cellular integration and restoration ofretinal structure) (MacLaren et al., 2006). Unfortunately, like RPCs,in humans these cells are isolated during late embryonic develop-ment, which raises serious ethical and practical concerns (i.e. itwould be difficult to obtain sufficient numbers of viable cells fortransplantation). Fortunately, both ESCs and iPSCs have all beenshown to give rise to photoreceptor precursor cells post-differentiation and following transplantation from new functionalphotoreceptors (Bartsch et al., 2008; Gonzalez-Cordero et al., 2013;Lamba et al., 2009; Tucker et al., 2013b, 2011a;West et al., 2012; Yaoet al., 2011). Of the differentiation paradigms used, ours wasdeveloped by combining aspects of previously published differen-tiation strategies (Ikeda et al., 2005; Lamba et al., 2006; Osakadaet al., 2008) which take into account the role of bone morpho-genic protein (BMP) and Wnt signaling pathway inhibition inneuroectodermal development (Anderson et al., 2002; Lamb et al.,1993; Mukhopadhyay et al., 2001), the role of IGF-1 in anteriorneural/eye field development (Pera et al., 2001), and Notchpathway inhibition in photoreceptor development (Jadhav et al.,2006). Using this differentiation program, iPSCs readily formembryoid bodies within days and early retinal progenitor cells

Fig. 6. IPSC-derived photoreceptors express photoreceptor-specific markers. A) ImmunofluiPSC-derived photoreceptor cultures. Inset shows a magnified view of recoverin-positive phthat is comprised of photoreceptor-like cells that express the photoreceptor-specific marke

(RPCs) within weeks. These iPSC-derived RPCs express the retinal-specific markers OTX2, SIX6, VSX2, RX and CRX. Following 2e3more months, these cells further differentiate into eyecup likestructures with post-mitotic photoreceptor precursor cells thatexpress the photoreceptor-specific markers recoverin andrhodopsin (Fig. 6), phosducin, red/green-opsin and blue-opsin(Tucker et al., 2013a,b, 2011a,b). Although promising forphotoreceptor-only diseases, the optimal developmental target isyet to be determined for RPE and choroidal endothelial cells. Assuch, in situations where multiple cell types are required furtherexperimentation is needed. In disease states such as these, autol-ogous cell sources will likely be critical.

Regardless of stem cell source and differentiation stage, differ-entiated retinal cell types will need to be purified from other celltypes present in the culture before transplantation in the clinic.Generating and purifying photoreceptors or choroidal endothelialcells in great enough numbers presents a challenge. Lamba andcolleagues purified photoreceptors generated from human iPS cellsusing fluorescence activated cell sorting (FACS) after transductionwith a lentivirus expressing GFP from a photoreceptor-specificregulatory elements. The purified photoreceptors integrated intomouse retina and expressed photoreceptor-specific markers Otx2,recoverin, and rhodopsin, supporting the proof-of-principle thatdifferentiated photoreceptor precursors can be purified andtransplanted successfully (Lamba et al., 2010). Genetically modifiedphotoreceptors, however, would not be desirable for treatment,thus alternate methods of purification should be explored.Lakowski et al. isolated photoreceptor precursor cells from devel-oping murine retinae using the photoreceptor cell surface antigensCd24a and Nt5e and FACS. The researchers transplanted theselected photoreceptor precursor cell population into wild-type ordegenerative murine host eyes (Crb1�/� or Prph2�/�). The selectedcells migrated into the outer nuclear layer, acquired photoreceptormorphology and expressed outer segment markers at an 18-foldhigher integration efficiency than that of unsorted cells (Lakowskiet al., 2011). These important experiments indicate the feasibilityof using cell selection strategies employing antibodies recognizingcell surface markers to purify photoreceptors for transplantation.One can envision a similar strategy for use with choroidal endo-thelial cells.

3) How will the local diseased environment respond to trans-planted 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.

orescent labeling of recoverin (green), tubulin (red) and DAPI (blue) in differentiatedotoreceptor-like cells. B) High magnification example of a differentiated neural rosetters, recoverin (green) and rhodopsin (red). Scale bar ¼ 400 mm.


It is believed that this “immune privilege” exists for the purpose ofprotection from unwanted inflammatory responses and helps tomaintain 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 Door-emaals observed that tumor cells injected into the anterior cham-ber of the eye survived, proliferated and formed intraocular tumors(Van Dooremaal, 1873). Many years later it was demonstrated thatthe eye is not entirely “immune privileged,” but the response tointraocular antigens is highly controlled and specialized, a phe-nomenon that became known as anterior chamber-associated im-mune deviation (ACAID) (Kaplan and Streilein, 1977; Stein-Streileinand Streilein, 2002; Streilein, 1987; Streilein and Niederkorn, 1981).ACAID is the process by which the eye communicates with thesurrounding immune system via the spleen to tolerate antigenpresenting cells in the eye and thus avoid a delayed, heightenedsystemic immune response (Stein-Streilein and Streilein, 2002;Streilein and Niederkorn, 1981). A common clinical example ofthe tolerance allowed due to ACAID is the allogenic transplantationof avascular corneal grafts, a highly successful surgery that requiresno immunological matching between donor and recipient, merelylocal immunomodulation via topical drops. Although the retinadoes not possess the same level of immune privilege as the anteriorchamber, the blood-retinal barrier (BRB) affords some protection.The BRB acts as a tight barrier between the retina and that of thesystemic circulation, namely circulating immunomodulatory andeffector leukocytes. The BRB actually consists of two separateanatomical sites: 1) the endothelial cells, pericytes, and glia of thenon-fenestrated inner retinal vasculature and 2) the tight junction-containing, monolayered RPE on Bruch's membrane between thefenestrated choroidal vasculature and outer retinal vasculature(Crane and Liversidge, 2008). In a normal, healthy retina, thoughnot immune privileged, the BRB provides the retina with an im-mune advantage, keeping it isolated from the surrounding immunesystem. However, when the retina is damaged, particularly if theintegrity of the RPE and choroidal vasculature is compromised,circulating immunomodulatory factors have easier access to theneural retina. In most degenerative retinal diseases, the RPE issignificantly injured. Using human donor eyes from patients withvarious retinal degenerative diseases like ABCA4-associated Star-gardt disease, Best disease and AMD, we have shown that loss ofRPE is a common characteristic among these disorders (Mullinset al., 2012, 2007). Support for the BRB playing a major role inconferring protection from the systemic immune system lies inevidence that the subretinal space becomes much more pro-inflammatory in the face of photoreceptor and RPE degeneration(Chinnery et al., 2012; Mullins et al., 2012; Rutar et al., 2010). There

Fig. 7. Cellular infiltrates in the vitreous cavity of a patient with CLN3-associated Batten diseassociated Batten disease showing bulls eye maculopathy and granularity of the fovea, boththe same eye in (A) and a magnified view of cellular infiltrates present throughout the vitr

are also reports that inflammation is heavily involved in the path-ogenesis of AMD (Ambati et al., 2013; Anand et al., 2003; Taralloet al., 2012; Whitcup et al., 2013). Furthermore, it has been sug-gested that the immune systemmay be critical in the pathogenesisof Batten disease. Cellular infiltrates have been observed in thevitreous cavity of molecularly-confirmed Batten patients (Fig. 7),and these patients also possess circulating autoantibodies, partic-ularly directed against GAD65 (Chattopadhyay et al., 2002; Dracket al., 2014; Pearce et al., 2004). Immunosuppressive agents havedemonstrated efficacy in slowing the progression of CLN3-associ-ated 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 numerousretinal degenerative diseases leaves the retina vulnerable tocirculating immune cells and immunomodulatory factors are thereason we believe that the most promising cellular population forfuture use in human cellular transplantation trials is that of autol-ogous, immunologically-matched patient-derived iPSCs. WhileESCs may be more readily available in a shorter timeframe, the factthat they are not immunologically matched between donor andrecipient makes it highly likely that they will incite an acute im-mune response, leading to rejection of the transplanted cells andperhaps further damage to an already diseased eye. This concern isof particular importance if the patient receiving ESCs still has somefunctional vision remaining. That being said, ESCs would likely stillbe a good option for transplantation and would be tolerated to abetter degree in early-stage diseased retinas in which the RPE isrelatively undamaged and BRB integrity remains intact. While us-ing autologous iPSCs will likely reduce the incidence of immuno-logical reactions and rejection of transplanted cells, the degree towhich immunologic matching will be required for the survival,integration, function, and longevity of stem cell-based retinaltransplants is a critical unanswered question in the field. Defini-tively testing this hypothesis will allow us to conserve societal re-sources (if patient-specific cell sources prove not to be required forsuccessful therapeutic transplantation); or, prevent harmful im-mune activation in visually impaired patients (if our studies showthat immune matching of the transplant is necessary).

4. Summary

Patient-specific induced pluripotent stem cells have emerged asa promising tool for identification and interrogation of disease-causing mutations, testing efficacy of novel therapeutics, and as acell source for autologous retinal cell replacement. As depicted inFig. 8, the key to patient-specific therapeutic development isthorough phenotypic assessment and accurate clinical diagnosis of

ase. A) Fundus photograph of the right eye of a child with molecularly-confirmed CLN3-common presentations in eyes afflicted with Batten disease. B) Slit lamp photograph ofeous cavity (outlined in red box).

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 familyhistory, high-resolution retinal imaging (OCT and fundoscopy), vi-sual field and electroretinographic analysis, followed by molecularconfirmation (genotyping) of disease-causing mutations viasequencing of patient DNA (from blood). A skin biopsy obtainedduring the initial clinical visit can be used for generation of autol-ogous patient-derived iPSCs. IPSCs and iPSC-derived retinal cellscan then be used to interrogate disease pathophysiology, developdrug, genome editing, gene augmentation and cell-based therapies,as well as to discover novel mutations that cannot be identifiedusing standard sequencing methods, such as those located in pro-moter or intronic regions of the gene. Finally, strategies developed

and tested against patient-specific disease mutations and pheno-types can then be utilized for human gene and cell replacementtherapies.

5. Future directions

Many exciting developments in the field of iPSC technologypresent an excellent opportunity to treat inherited retinal degen-erative diseases and ultimately improve patients' lives. IPSCs can beemployed to investigate and determine disease mechanisms fromindividual patients as well as determining pathogenicity of novelmutations. Knowledge gained from these experiments can be used


to develop and test drug- and gene-based therapies. Trans-plantation of gene-corrected patient-specific iPSC-derived retinalneurons will treat individuals with advanced-stage retinal degen-erative disease. These approaches will be expedited through pro-duction of clinical-grade reagents (i.e. patient-specific iPSC-derivedretinal photoreceptor cell lines and viral vectors) in dedicatedcurrent good manufacturing production (cGMP) facilities. Clinical-grade stem cells and therapeutic vectors can be utilized in acompassionate use manner to treat aggressive neurodegenerativeblinding diseases to pave the way for treatments of other rareinherited retinal dystrophies that fall below the commercialviability 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 orga-nizations: NIH Directors New Innovator Award 1-DP2-OD007483-01; NEI EY017451; HHMI; Foundation Fighting Blindness; StephenA. Wynn Foundation; Grousbeck Family Foundation; Leo, Jacques&Marion Hauser Family Vision Restoration Fund; NIH F32 EY022834.

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http://dx.doi.org/10.1086/431426

http://dx.doi.org/10.1186/1471-2350-9-51


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