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DOI: 10.1167/tvst.4.6.2

Article

Report on the National Eye Institute Audacious GoalsInitiative: Photoreceptor Regeneration and IntegrationWorkshop

David M. Gamm1, Rachel Wong2, and the AGI Workshop Panelists*1 Department of Ophthalmology and Visual Sciences and McPherson Eye Research Institute, University of Wisconsin-Madison, Madison,WI, USA2 Department of Biological Structure, University of Washington, Seattle, WA, USA

Correspondence: David M. Gamm,Department of Ophthalmology andVisual Sciences and McPherson EyeResearch Institute, University of Wis-consin-Madison, Madison, WI, 53705,USA. e-mail: [email protected]

Received: 9 October 2015Accepted: 15 October 2015Published: 17 November 2015

Keywords: photoreceptors; regen-erative medicine; stem cell

Citation: Gamm DM, Wong R, AGIWorkshop Panelists. Report on theNational Eye Institute AudaciousGoals Initiative: Photoreceptor Re-generation and Integration Work-shop. Trans Vis Tech. 2015;4(6):2,doi:10.1167/tvst.4.6.2

The National Eye Institute (NEI) hosted a workshop on May 2, 2015, as part of theAudacious Goals Initiative (AGI) to foster a concerted effort to develop novel therapiesfor outer retinal diseases. The central goal of this initiative is to ‘‘demonstrate by 2025the restoration of usable vision in humans through the regeneration of neurons andneural connections in the eye and visual system.’’ More specifically, the AGI identifiedtwo neural retinal cell classes—ganglion cells and photoreceptors—as challenging,high impact targets for these efforts. A prior workshop and subsequent white paperprovided a foundation to begin addressing issues regarding optic nerve regeneration,whereas the major objective of the May 2015 workshop was to review progresstoward photoreceptor replacement and identify research gaps and barriers that arelimiting advancement of the field. The present report summarizes that discussion andinput, which was gathered from a panel of distinguished basic science and clinicalinvestigators with diverse technical expertise and experience with different modelsystems. Four broad discussion categories were put forth during the workshop, eachaddressing a critical area of need in the pursuit of functional photoreceptorregeneration: (1) cell sources for photoreceptor regeneration, (2) cell delivery and/orintegration, (3) outcome assessment, and (4) preclinical models and target patientpopulations. For each category, multiple challenges and opportunities for researchdiscovery and tool production were identified and vetted. The present reportsummarizes the dialogue that took place and seeks to encourage continuedinteractions within the vision science community on this topic. It also serves as aguide for funding to support the pursuit of cell and circuit repair in diseases leading tophotoreceptor degeneration.

Introduction

It has long been a goal of vision science to restoresight in patients blinded by the physical loss ofphotoreceptors, either as a result of degenerativedisease or injury. However, until recently the toolsand knowledge required to achieve this goal havebeen limited. With the advent of pluripotent stem celltechnology and in situ reprogramming techniques, aswell as devices to assess cell structure and function invivo, we now have an opportunity to devise and testphotoreceptor regeneration strategies in a moredirected and rigorous manner. In addition, anincreasing willingness to share expertise and resources

among scientists across disciplines has provided anenvironment that favors research acceleration overindividual accomplishment. Such integrated researchefforts should lead to the elucidation of cell andmolecular mechanisms that promote or hinderphotoreceptor regeneration, allowing us to buildupon successes and overcome failures in an expeditedmanner.

When contemplating the prospect of photorecep-tor regeneration, it is helpful to start with a set ofbasic assumptions. The first, and perhaps mostfundamental, is that meaningful restoration of visionvia photoreceptor regeneration is attainable in hu-mans. However, defining ‘‘meaningful’’ vision im-

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provement will require careful and inclusive debate inorder to set realistic expectations and avoid misun-derstandings among scientists, clinicians, patients,and other vested individuals when assessing theprogress and impact of the Audacious Goals Initiative(AGI). Second, additional knowledge and technolog-ical advancements are still needed to confidentlyproceed with photoreceptor regeneration therapies,although it can be argued that such confidence mayonly come with carefully designed human clinicaltrials. Other safe assumptions include: no singletherapeutic approach will succeed under all condi-tions of photoreceptor loss, treatment effects willlikely be modest (at least initially), and even successfulstrategies will need refinement beyond the 2025 goalfor the AGI. Lastly, paramount to all other aspects ofthis initiative is the need to maximize patient safety.With these thoughts in mind, workshop participantssought to delineate the research challenges andknowledge gaps that need to be addressed toaccomplish the goal of photoreceptor regenerationin humans.

Cell Sources for Photoreceptor Regeneration

Photoreceptor replacement can theoretically bepursued using either exogenous or endogenous cellsources, with both approaches potentially employinga variety of donor cell types. For exogenousphotoreceptor replacement, possible donor sourcesinclude human embryonic or induced pluripotentstem cells (hESCs or hiPSCs), prenatal retinal cells,cultured adult tissue stem cells, or transdifferentiatedcell cultures. Endogenous repair strategies couldtarget host Muller glia, retinal pigment epithelium(RPE), or perhaps ciliary margin cells, although thelast option was felt to have limited appeal in part dueto their localization to the far retinal periphery.

Human pluripotent stem cells were felt to be themost promising donor source for exogenous photo-receptor replacement given their capacity to producecone and rod precursors as well as more maturephotoreceptor progeny.1–14 Furthermore, evidencesuggests that photoreceptor lineage cells derived frompluripotent stem cell sources can integrate and restorevisual function in at least some mouse models ofphotoreceptor dysfunction.13–17 Identified challengesto the use of hESCs and hiPSCs as sources forexogenous photoreceptor regeneration include: (1) theinherent heterogeneity of differentiating neural retinalcell populations, (2) the potential for donor cellrejection, (3) difficulties adapting lab-based protocolsto current Good Manufacturing Practice (cGMP)

guidelines, (4) the variability of photoreceptor cellproduction across lines, (5) enrichment of photore-ceptor lineage cells, (6) the high cost and lengthy timerequirements of cell manufacture, and (7) assuringpatient safety. Between hESCs and hiPSCs, the latterwas noted to have an advantage with regard towidespread ethical acceptance and the theoreticalpotential to circumvent immune rejection via autol-ogous cell transplantation. That being said, autolo-gous therapies carry a huge cost and time burden andmight not be necessary, particularly considering thefuture availability of banks of homozygous HLA‘‘super donor’’ hiPSC lines18–22 or genetically engi-neered ‘‘immuno-neutral’’ hESCs.

Advantages and disadvantages of 2D versus 3DhESC and hiPSC retinal differentiation protocolswere discussed, the former being more conducive toscale up but perhaps less capable or efficient atproducing integration-competent photoreceptor pre-cursors.16,17 Participants acknowledged the need toenrich for hESC- and hiPSC-derived photoreceptorcells prior to transplantation, but it was unclear whatlevel of purity was optimal or necessary to promotesurvival and integration. Enrichment can be achievedin mouse ESC cultures using cell surface markers toselect integration-competent photoreceptor precur-sors23; however, this strategy must be confirmed withhESCs and hiPSCs. Also imperative are studies tobetter understand the diversity of photoreceptor celltypes and maturation states in these cultures, which isessential for the interpretation and subsequent refine-ment of transplantation strategies. The specific needto concentrate on the production of cones was voicedby numerous workshop participants, owing to theirunique importance to human visual function. Lastly,concerns regarding the potential for teratoma forma-tion from residual pluripotent cell contamination indonor cell populations were raised, although it wasalso pointed out that methods to detect even minuteamounts of unwanted cell types are available.

The use of primary prenatal tissue and thepotential to transdifferentiate nonphotoreceptor cellsinto cone and/or rods (or rods into cones) were alsodiscussed. It was acknowledged that these approachescarry some benefits, including less time required togenerate donor cells and likely lower costs as well.However, ethical concerns surrounding the formercell source and the degree of authenticity achievablewith transdifferentiation were among issues thatreduced enthusiasm for these options.

Numerous advancements in endogenous replace-ment of neural retinal cell types have been made


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recently, building on decades of ground-breakingresearch performed in fish, amphibians, and chicks,which display varying amounts of retinal regenerativecapacity.24–28 Conversion of Muller glia to retinalinterneurons in damaged mammalian retina has beendemonstrated via introduction of the proneuraltranscription factor Ascl1,29 and newts display robustendogenous retinal repair originating from RPE cellsfollowing injury.30 The existence of a quiescentpopulation of RPE stem cells in adult human retinaoffers another population of endogenous cell typesthat theoretically could be commandeered to trans-differentiate and replace lost photoreceptor cells.31,32

However, major challenges to endogenous photore-ceptor regeneration remain to be addressed, mostnotably the fact that significant production ofphotoreceptors has not yet been demonstrated usingthis approach. Furthermore, the conversion of largenumbers of Muller glia to retinal neurons couldoverdeplete this important cell type. Even when thishurdle is overcome, it is unclear whether the newlyconverted photoreceptors will be appropriately posi-tioned within the retina, or if their location evenmatters as long as they are properly connected withinthe retinal circuitry. At the end of this discussion, itwas largely felt by workshop attendees that, althoughendogenous photoreceptor regeneration has a highceiling in terms of restoration of visual function,exogenous strategies are further along at present andmore likely to move toward clinical trials in the nearterm.

Delivery and Integration

Workshop participants were asked to discuss thekey steps that are required to optimize the introduc-tion of donor cells into the host retina. Two areaswere identified as particularly challenging: (1) im-proving the survival rate of photoreceptor transplan-tation, and (2) promoting synaptogenesis oftransplanted cells to restore light-driven signals.

There are currently three major strategies for celldelivery: subretinal injections of dissociated cells,retinal tissue transplant, and introduction of cellsseeded onto engineered scaffolds. Discussants agreedthat progress has been made for all approaches, but itis clear that no single approach has as yet overcomeall of its challenges.16,33–38 For example, whereas cellsurvival rates are better in tissue transplants, subret-inal delivery of dissociated photoreceptors has led tomore consistent functional integration.39 Overall,attendees favored exogenous photoreceptor deliveryusing stem-cell derived donor sources, but clear

arguments were made for endogenous production ofphotoreceptors via delivery of reprogramming vectorsto host cell targets such as Muller glial or RPE.Regardless of the strategy, it was the consensus of thegroup that all delivery methods need to be improvedin order to provide the number of integrated orreprogrammed cells necessary for significant visionrestoration.

The discussion then focused on the important issueof cone replacement, for which little information iscurrently available. Survival and integration rates ofrod photoreceptors in rodent models have improved,and levels of cell and circuit function can be achievedto the extent that visual behavior is partially regainedusing this photoreceptor subtype.16,33,40–42 In con-trast, attempts at cone photoreceptor replacementtherapy has thus far only achieved low levels ofintegrated cones.43,44 What factors underlie thisdiscrepancy? First, it remains challenging to produceand deliver large quantities of cones from donor tissueor stem cell cultures, where they constitute a minorityphotoreceptor subpopulation. A second issue is theapparent poor long-term survival of transplantedcones, even after they integrate into the host retina.44

Lastly, the diverse and complex circuits formedbetween cones and their postsynaptic cells make itmore demanding to obtain an accurate assessment ofthe extent of cone integration at the circuit level.Discussants raised the need to improve our under-standing of cone photoreceptor biology, function, andrepair, particularly in relation to the fovea in humansand nonhuman primates.

A second major area that requires considerableattention is the promotion of synaptogenesis betweentransplanted photoreceptors and host bipolar cells inthe degenerating retina. It is widely recognized thatgliosis and remodeling of the diseased retina seriouslyimpair photoreceptor integration. However, an un-derstanding of how these disruptive events aretriggered and subsequently evolve during diseaseprogression is still lacking. Increasing this knowledgeis critical for overcoming hurdles imposed by a hostilehost environment that undoubtedly imposes limits tophotoreceptor survival and integration.

Outcome Assessment

Workshop participants were asked the followingquestions regarding outcome assessment: (1) How cantransplanted photoreceptors be distinguished fromhost cells? (2) What technology (not currentlyavailable) is most needed to ensure photoreceptorreplacement and functional integration in patients?


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(3) What are relevant anatomical and functionaloutcomes?

To date, these questions have been addressed inanimal models using a large battery of in vitro and invivo assays, ranging from morphological analyses oftransplanted photoreceptors and their synapses tofunctional recordings including electrophysiology,electroretinography (ERG), and measurements ofpupillary light reflexes and light responses in highervisual centers.13,39 Moreover, several behavioralassays have been devised to test restoration of motionand contrast detection in rodents.39 Workshopparticipants indicated that one of the roadblocks thatmost severely limits translation of work in animalmodels to humans is a general lack of imaging toolsfor assessing photoreceptor survival and integrationin living patients. Much of the discussion centered onthe need to develop such technology specifically forhumans. The importance of standardizing assaysacross patient populations and test sites was alsoraised.

With regard to outcome assessments in humanpatients, participants felt there was a need to developnew tools to assess visual function for photoreceptorregeneration trials. Such a need has been underscoredin RPE65 gene therapy trials for Leber congenitalamaurosis, in which patients showed significantimprovements in visual behavior without documentedchanges in vision assessment metrics traditionallyused in clinical trials (e.g., Snellen acuity).45,46

Preclinical Models and Target PatientPopulations

Numerous options for preclinical model systemsare available to investigators, including small andlarge animals, nonhuman primates, and even cellculture systems. Critical questions regarding preclin-ical safety and efficacy studies for photoreceptorregeneration include: (1) What will the Food andDrug Administration (FDA) require? (2) What arethe expectations of the scientific and clinical commu-nities and the public? (3) What useful information canwe hope to obtain prior to human clinical trials? Todate, the vast majority of in vivo experimentspertaining to exogenous photoreceptor regenerationhave been performed in mice using allografts.Furthermore, numerous mouse models of photore-ceptor degeneration have been employed with vari-able results. These important efforts have revealedthat certain mouse models are more amenable todonor photoreceptor integration than others, likely

due to the relative absence of physical barriers withinthe recipient tissue (e.g., gliotic scar, intact outerlimiting membrane).40 Therefore, it is possible thatthat the structural state of the host retina, and notnecessarily the cause of the disease or injury, is theprimary dictator of the success or failure of photore-ceptor replacement therapies. Mice have also beenused to show proof of concept for endogenous retinalneuron regeneration in mammals. Similar to exoge-nous replacement approaches, endogenous photore-ceptor regeneration may ultimately be limited by hostretinal structure and the degree of scar formation andremodeling present at the time of intervention.

A major concern set forth by the discussants forboth exogenous and endogenous regeneration strate-gies was the relative reliance of the field on mousemodels to demonstrate safety and efficacy. Humancells in general show very poor survival as xenografts,and the behavior of human donor photoreceptor cellsin mouse (or other animal models) may inadequatelyinform or perhaps even mislead investigators orregulatory agencies in their quest to predict patientresponses to therapeutic interventions. One proposedsolution was to move more quickly toward nonhumanprimate studies. However, the use of macaques andlarger monkeys is costly, time consuming, and notpractical for researchers at most institutions. Thus,there was a perceived need to develop and provideaccess to colonies of smaller monkey species toinvestigators, and to devise means to simulateconditions of photoreceptor loss in these animals.

Finally, workshop participants discussed factorsthat might go into choosing a first indication forphotoreceptor regeneration therapies. Monogenetic,early-onset photoreceptor degenerative processes of-fer a more predictable disease course than most late-onset disorders with complex genetics, but candidatesin the former category are rare, and industry partnersmay not be as interested in supporting orphan diseasetrials. Interestingly, it was felt by many that the stageof disease and/or the structural condition of the retinawas as important (or more important) in choosingpatients than the underlying cause of the photorecep-tor loss for two major reasons. First, the early andmid-stages of disease in which a significant number offunctional photoreceptors still remain are unlikely tobe first targets for cell regeneration therapies. Indeed,at these stages, initial therapeutic efforts are probablybetter directed toward cell preservation or genereplacement, if feasible. Eventually, as the risk/benefitratio of photoreceptor regeneration approaches be-comes clearer, intervention at earlier disease stages


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may be deemed appropriate. Second, very late stagesof disease or injury may be accompanied by pervasivehost retinal remodeling and gliosis, creating a ‘‘pointof no return’’ for regeneration of a single retinal celltype. As a result, optimizing the timing of photore-ceptor replacement is almost assuredly going torequire advanced imaging and functional testingpre-operatively to predict the degree of ‘‘meaningful’’vision restoration possible in a given patient candi-date.

Gaps in Scientific Knowledge and

Barriers to Progress

Category 1: Cell Sources for PhotoreceptorRegeneration

Topics discussed under this category included (1)exogenous versus endogenous photoreceptor regener-ation strategies, (2) rod versus cone production, (3)demonstration of donor cell authenticity, and (4)safety considerations.

Exogenous Versus Endogenous Cell SourcesTwo major approaches to photoreceptor regener-

ation were evaluated: (1) photoreceptor replacementvia transplantation of exogenous donor cells, and (2)activation and/or reprogramming of endogenousretinal cell types to produce new photoreceptors. Amajor gap identified for the exogenous approach wasa relative lack of detailed investigation into thedynamic and heterogeneous composition of retinal(and nonretinal) cells present in differentiating hESCsand hiPSCs, as well as primary and expanded humanprenatal retinal cultures. Such knowledge wouldprovide a basis for evaluating why and how donorcell integration occurs, given current evidence thatonly a subset of photoreceptor subtypes and differ-entiation stages may be competent to migrate intohost retina and establish functional synapses. For theendogenous regeneration approach, identification ofconditions and factors specifically necessary forconversion of Muller glia and RPE (and perhapsother neural retinal cell classes) to photoreceptors—and cones in particular—is a high priority. Inaddition, methods to control the localization of newlyreprogrammed cells and encourage their synapticintegration are necessary to move the research towardclinical trials. To address these issues, the groupidentified efforts aimed at understanding mechanismsof endogenous regeneration in fish, amphibians, andchick as being highly important.

Rod Versus Cone ProductionConditions favoring rod precursor integration

have been examined in considerable detail in themouse, but efforts to promote cone replacement havebeen much less successful to date. Given that ourmost critical visual functions rely on cones, thepursuit of methods to enrich for their productionfrom exogenous and endogenous sources (with anemphasis on medium and long wavelength cones) wasthought to be a high priority area for the AGI.However, rod replacement may be more attainable inthe short term given current animal model data andthe preponderance of rod production over cones innormal human retinogenesis. Another advantage ofpursuing rod replacement is that earlier disease stagescan be targeted, which may lead to improved rodintegration and function while also serving to protectremaining foveal cones. However, rod productioncurrently takes an exceptionally long time in hESCand hiPSC cultures, and much greater cell quantitiesand host coverage areas would likely be needed whencompared to cone replacement strategies. Regardlessof the preferred donor photoreceptor subtype, currentproduction protocols yield a mixture of photorecep-tor cells with varying maturation states, along withnonphotoreceptor cell types. As such, developingmethods to modulate cell composition and aligndifferentiation states in vitro was also considerednecessary to accelerate progress.

Demonstration of New Photoreceptor Authenticityand Functional Capacity

In addition to probing the composition of celltypes produced from pluripotent stem cells, prenataltissue, or reprogrammed cells, efforts to understandphotoreceptor differentiation and transdifferentiationmechanisms and to overcome limitations regardingtheir functional potential were deemed critical forfuture study, for reasons discussed previously.

Understanding the Role of Muller Glia in Health andDisease

Members of the workshop pointed out theimportance of Muller glia in maintaining a healthyenvironment for new photoreceptors, as well as theiropposing potential to create significant physicalbarriers to successful photoreceptor regeneration.Further work was encouraged to define the triggersthat tilt the balance toward induction of deleteriousMuller glia responses in an effort to mitigate orreverse these effects. Successful pursuit of this goalwould aid both exogenous and endogenous regener-ation approaches, perhaps more so in the latter since


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Muller glia are a major target for endogenousreprogramming. Studies to determine whether distinctsubclasses of Muller glia cells exist that are more orless capable of transdifferentiation or gliosis was alsoidentified as a worthwhile component of the AGI. Ofnote, such a situation exists in adult human RPE,which is known to possess a subpopulation thatretains quiescent stem-like properties that predisposesit to transdifferentiation.31,32 Thus, investigation intothe unique characteristics and capabilities of thisadult ‘‘RPE stem cell’’ was also deemed important inorder to determine whether it can be harnessed forphotoreceptor protection and/or regeneration pur-poses.

Safety ConsiderationsSafety issues for exogenous photoreceptor replace-

ment therapies that workshop participants felt neededto be addressed included induction of inflammationand gliosis, host immune rejection, dedifferentiationof donor cells in vivo, and the potential for prolongedor recurrent retinal detachment. Endogenous photo-receptor regeneration strategies raised concerns forthe possibility of differentitation to unwanted celltypes, enhancement of gliosis due to Muller gliaactivation, and deleterious or short-lived effects ofviral vectors. For both approaches there wereconcerns for enhanced degeneration of host retina,tumorigenicity, establishment of inappropriate syn-aptic connections, and poor long-term viability andfunction of new photoreceptors within a diseased,damaged, and/or toxic host tissue environment.

Category 2: Delivery and Integration

The discussion centered on two major areas: (1)how to improve the number of photoreceptors thatsurvive and integrate, and (2) how to promote theirsynaptogenesis to recreate circuits that can restoremotion detection, visual acuity, contrast sensitivity,and color vision.

Improving the Number of Photoreceptors ThatSurvive and Integrate Into Host Retina

It was suggested that an immediate goal should beto ascertain how many photoreceptors (rods or cones)are needed to integrate successfully in order to restorevisual function. In mice, rod delivery and integrationappear sufficient to restore visual behavior, but conephotoreceptor replacement and synaptic integrationremains immensely challenging. Several discussantssuggested that one way to overcome limitations incone replacement therapy is to better understand the

cellular and molecular factors that specify conephotoreceptor fate during development, especially inthe human retina. This knowledge could then be usedto promote cone generation either in vitro or in situ.However, it was also recognized that cones can varyin morphology and possibly function across theretina. Thus, defining differences in the morphology,molecular make-up, and connectivity of foveal versusperipheral cones in humans and nonhuman primatesrequires further investigation. Finally, current ap-proaches for photoreceptor replacement center largelyon rodent models, which are not ideal for investigat-ing cone replacement therapies. Thus, one importantconsideration for the future is to determine whichsmall and large animal model systems are best suitedfor testing strategies for cone replacement therapy.

Photoreceptor Synaptogenesis and Wiring SpecificityUltrastructural studies to date demonstrate that

transplanted rod photoreceptors connect with rodbipolar cells. There is evidence for cone synaptogen-esis in mouse models of cone replacement, but thespecificity of these connections remains unknown.The discussants felt that the field lacks a basicknowledge of the molecular cues and cellular interac-tions that regulate specificity in the synaptic connec-tivity of both rods and cones during normaldevelopment. Filling this knowledge gap could helpidentify factors that could be employed to promoteappropriate wiring of transplanted rod or conephotoreceptors. Moreover, knowledge of the mecha-nisms underlying photoreceptor circuit developmentcould facilitate the design of strategies to redirectconnections of surviving cells and restore vision vianovel circuits. For example, the discussants raised thepossibility that cone-driven signals could be restoredby manipulating opsin expression in surviving rodphotoreceptors (i.e., turn rods into cones). However,there was debate as to whether this approach wouldwork since the downstream signaling cascades aredifferent for rods and cones.

Remodeling of the Diseased EnvironmentThere was general agreement that a major road-

block in rebuilding circuitry in the damaged retina isthe aberrant remodeling that occurs after photore-ceptor loss.47,48 We currently do not know how toprevent such remodeling, since the factors and cellularinteractions that cause retinal neurons to alter theirposition, morphology, connectivity, and functionhave remained elusive. It is clear, however, thatreactive gliosis (especially gliosis involving Mullerglial cells) can present a major physical barrier to


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photoreceptor integration and play a role in theremodeling process. Thus, increasing our understand-ing of the intrinsic and extrinsic controls over gliosis isimmensely important. The discussants emphasized aneed to discover strategies to prevent gliosis as well asto direct glial cells along paths that could actually bebeneficial, such as production of trophic factors or astargets for endogenous repair. Furthering knowledgeof the factors that control reprogramming in zebra-fish,24,25 which leads to the production of both Mullerglial cells and neuronal progenitors, would also helpinform the field how to replenish lost photoreceptorswithout excessively depleting the retina of these glia.There was also consensus that we need to betterunderstand the role of microglia in the remodelingprocess, as well as the innate immune response.

Because remodeling is a progressive process withinthe diseased retina, the group discussed the impor-tance of intervening at early stages of disease to takeadvantage of the more conducive environment forphotoreceptor integration and circuit rewiring. Sinceremodeling appears to accelerate with cone degener-ation, one unconventional approach that was sug-gested was to transplant cones specifically to halt orslow down retinal remodeling, thereby providingmore time to develop a more definitive treatment.

Where Do We Set the Bar for PhotoreceptorIntegration?

It was also recognized that the field shouldprioritize its goals—for example, should we aim forrestoring high acuity vision, a large visual field, and/or cone-mediated vision? It was argued that patientswithout any visual function would benefit immenselyfrom the restoration of any visual function. Becauserods may integrate more readily than cones, it wassuggested that even placing rods in the fovea mightprovide some improvement in visual function if theysucceeded in connecting with cone bipolar cells.Finally, further thought should be given to combiningphotoreceptor replacement therapy with retinal pros-thetic technology to enhance the outcome of thelatter.

Category 3: Outcome Assessment

Currently, one of the most significant challengesfor photoreceptor regeneration is the lack of technol-ogy to enable visualization of transplanted photore-ceptors in patients. Devising such tools is critical formonitoring the survival, location, and functionalimpact of cell integration in clinical trials. Muchprogress has been made in the development of

imaging methods that use adaptive optics to visualizefluorescently tagged cells in nonhuman primates.Discussants felt that new fluorescent tags are neededthat label transplanted photoreceptors without mod-ifying their differentiation and function. It was alsonoted that novel split detector technology is availableto visualize inner segments of photoreceptors inhumans, thereby bypassing the need to label thecells.49,50 However, there is concern that such imagingapproaches will not work in severely degeneratedretinas where the orderly arrangement of photorecep-tors is disrupted. Thus, there is a pressing need todevelop innovative approaches to visualize andmonitor photoreceptor transplants, and to test themfirst in nonhuman primate to assess their utility infuture clinical trials.

Category 4: Preclinical Models and TargetPatient Populations

Knowledge gaps and hurdles pertaining to thisdiscussion category were prevalent and challengingfor participants to devise solutions to overcome. Itwas also noted that choices regarding preclinicalmodels and target patient populations cannot bemade in isolation, since they require knowledge of thedonor cell population and other variables presented inthe preceding sections. However, notable difficultiessurrounding preclinical model selection include (1)limited confidence in extrapolating safety and efficacyresults from mouse models to future human clinicaltrials, (2) a relative lack of animal models of selectivecone loss, and (3) a poor understanding of FDArequirements for proceeding with phase I trials for cellreplacement therapies. While nonhuman primateswere felt to be anatomically far superior as a modelsystem for cone and rod replacement, no such modelsof degenerative photoreceptor diseases are available,limiting their utility. The excessive cost and restrictedavailability of nonhuman primates also poses aconsiderable hurdle to their widespread use.

Challenges to selecting initial disease targets forphotoreceptor regeneration therapies emanate fromlimitations in our understanding of genetic, epigenet-ic, and biological changes that occur during retinaldisease and injury. Further hindrances come fromdifficulties assessing host retinal structure and func-tion on a cellular level in vivo, which is needed toestimate the presence and degree of glial scarring andinner retinal remodeling. Better visualization tech-niques are also needed to define the course and impactof host inflammation on donor cell survival and


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integration. The workshop participants acknowledgedthe recent AGI awards directed toward advancingocular imaging technology as taking a significant steptoward addressing these gaps. Further investment inthe study of the longitudinal courses of outer retinaldegenerative diseases will also be helpful in thisregard.

Areas for Open-Ended, Non-

Hypothesis-Driven Discovery Research

Cell Sources� For exogenous photoreceptor regeneration effortsto advance and succeed, knowledge of the compo-sition and maturation states of differentiatingdonor cell populations (particularly photoreceptorsubtypes) is required. Methods to modulate cellularheterogeneity and enrich for particular photorecep-tor cell types are also of high value.

� New cell surface markers or other tools are neededto distinguish and separate human photoreceptorsfrom other retinal cell classes (or enrich for conesubtypes versus rods).

� In the pursuit of endogenous photoreceptor regen-eration, research is needed to understand mamma-lian Muller glia development and probe theirpotential to revert to a progenitor state. Suchefforts would benefit from continued examinationof classic lower vertebrate models capable ofregenerating photoreceptors from Muller glia, inorder to understand why that capacity is lost inmammals. Similarly, projects aimed at probing thepotential for mammalian RPE or other retinal celltypes (e.g., bipolar cells) to transdifferentiate andregenerate photoreceptors are of interest. Also, thepossibility that specific subpopulations of Mullerglia and RPE cells exist that have quiescentmultipotent retinal progenitor properties shouldbe examined. Lastly, efforts to bring aboutendogenous photoreceptor regeneration in mam-mals would be greatly aided by a better under-standing of retinal repair mechanisms exhibited byteleosts, urodeles, and chicks.

� Both exogenous and endogenous photoreceptorrepair strategies would benefit from an improvedunderstanding of how retinal progenitor cellsdifferentiate into neural retinal cell classes, includ-ing photoreceptor subtypes. This is particularlyimportant to investigate in donor cell populationssuch as hESCs and hiPSCs.

Reassembling Photoreceptor Circuits� Discovery science aimed at increasing our under-standing of the molecular and cellular mechanismsthat control photoreceptor fate and differentiationduring normal development should be encouraged.Especially important is increasing this knowledgefor cone photoreceptors, particularly in humansand nonhuman primates.

� Innovative approaches that prevent or slow downdegeneration, gliosis, and remodeling are needed tocreate a more conducive environment for synapto-genesis of transplanted photoreceptors. As such,factors that control the responses of Muller glialcells and microglia cells upon injury and in earlystages of the diseases need to be determined.

� Increasing our understanding of the cellular andmolecular mechanisms that regulate photoreceptorsynaptogenesis and circuit development in vivo alsoneeds to be advanced. Such knowledge will greatlyfacilitate the design of knowledge-based strategiesthat drive photoreceptor circuit repair in animalmodels and eventually, in patients.

Assessing Outcomes� It remains challenging to track the survival oftransplants in a human eye and to monitor theneural signals generated by their connectivity. Toovercome these hurdles, we need to devise betterways of introducing markers into the human eye.Innovations in optical coherence tomography(OCT), adaptive optics, and techniques that inte-grate existing approaches are rapidly advancing,but as yet there is no effective approach to evaluatethe in vivo performance of transplanted cells. Thus,an important goal in this category is to developmethods to distinguish newly introduced cells andsurviving host cells.

� While fluorescent markers have been used success-fully in animal models and remain a viable optionto track donor cells, the field should also invest indiscovering label-free imaging techniques for use inhumans.

� Better methods are needed for assessing thefunctional response of integrated cells and identi-fying whether these cells have made meaningfulconnections that directly lead to improved visualbehavior in patients.

Preclinical Models and Target PatientPopulations� Development of affordable and convenient nonhu-man primate models is a worthwhile goal in order


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Box 1. Critical questions to address in order to accelerate progress toward functional photoreceptor regeneration.


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to more accurately assess regeneration and/orreplacement of cones within the macula and fovea.In addition, effective and reproducible means ofinducing localized photoreceptor degeneration insuch models is important to provide a platform forassessing restoration of cell circuitry and visualfunction.

� Critical evaluation of the utility and predictivepower of widely employed mammalian animalmodels of retinal degeneration (e.g., mouse models)is required to assure their appropriate selection anduse. Special attention should be given to developingbetter age-related macular degeneration models.

� The possibility of making in vitro models of normaland diseased human retina from hESCs, hiPSCs,and/or prenatal tissue should be investigated. Forexample, hiPSCs could be used to create three-dimensional disease-in-a-dish models to test func-tional photoreceptor replacement and/or regenera-tion (e.g., examination of Muller glia plasticity orphotoreceptor synaptogenesis).

� Further investigation of lower vertebrate models ofadult photoreceptor regeneration is warranted togain further insights into the biology of regenera-tion and ascertain why mammalian Muller glia andRPE cells lose this capacity.

� Studies should be solicited to determine what typesand stages of diseases would be best to target ininitial photoreceptor regeneration trials.

� Involvement of ocular imaging specialists is neededto ensure that future clinical trials will have a highlikelihood of yielding reliable structure/functioninformation in living patients.

Summary

Regeneration of photoreceptors in patients withsevere vision loss due to outer retinal degeneration is atruly audacious goal, but one that is now conceivableowing to recent scientific advancements superimposedon over a century of research progress. The eye isuniquely suited to test strategies to replace neurons,and photoreceptors are particularly attractive asdonor cells given their short-ranged connections andthe surgical accessibility of the outer retina. Still,much work is still required to realize this goal, both interms of acquisition of basic scientific knowledge anddevelopment and refinement of critical technology.Such advancements should improve our ability togenerate ‘‘replacement’’ photoreceptors and assesstheir ability to integrate and restore host retinal

circuitry and function. Team-based research and opensharing of results will aid these efforts and helpovercome the formidable hurdles that lie ahead.Managing expectations is also important, since resultsfrom early patient trials are likely to be modest due toa necessary focus on safety. However, improvementsin clinical trial design should allow us to learn fromour successes and failures and continuously improvestrategies to regenerate photoreceptors, and in sodoing provide tangible results to a wide range ofpatients who currently have no treatment options.

Acknowledgments

Disclosure: D.M. Gamm, None; R. Wong, None

* Additional workshop panelists (affiliations listedin Appendix A): Kapil Bharti (National Eye Insti-tute), Seth Blackshaw (Johns Hopkins University),Mark Blumenkranz (Stanford University), MariaValeria Canto-Soler (Johns Hopkins University),Ching-Kang Jason Chen (Baylor College of Medi-cine), Dennis Clegg (UC Santa Barbara), Katia DelRio-Tsonis (Miami University of Ohio), John Dow-ling (Harvard University), Jacque Duncan (UC SanFrancisco), Wei Li (National Eye Institute), PamelaRaymond (University of Michigan), Thomas Reh(University of Washington), Austin Roorda (UCBerkeley), Josh Sanes (Harvard University), PaulSieving (National Eye Institute), Jane Sowden (Uni-versity College London), Masayo Takahashi (RIKENCenter for Developmental Biology), Sally Temple(Neural Stem Cell Institute), Budd Tucker (Universityof Iowa), Monica Vetter (University of Utah), Shu-Zhen Wang (University of Alabama at Birmingham),Ron Wen (Bascom Palmer Eye Institute), DavidWilliams (University of Rochester), Wai T. Wong(National Eye Institute), Ting Xie (Stowers Institutefor Medical Research), Kang Zhang (UC San Diego).

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Appendix A

Kapil Bharti, PhDEarl Stadtman Tenure-track InvestigatorNational Eye InstituteNational Institutes of [email protected]

Seth Blackshaw, PhDAssociate Professor of NeuroscienceJohns Hopkins University School of [email protected]


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Mark Blumenkranz, MDH.J. Smead Professor and ChairDepartment of OphthalmologyStanford [email protected]

Maria Valeria Canto-Soler, PhDAssistant Professor of OphthalmologyDepartment of OphthalmologyJohns Hopkins [email protected]

Ching-Kang Jason Chen, PhDAlex R. McPherson Retinal Research FoundationEndowed ChairProfessor of OphthalmologyBaylor College of [email protected]

Dennis G. Clegg, PhDProfessor and Co-DirectorCenter for Stem Cell Biology and EngineeringUniversity of California Santa [email protected]

Katia Del Rio-Tsonis, PhDProfessor of BiologyMiami [email protected]

John E. Dowling, PhD, A.B.Gordon and Llura Gund Professor of NeurosciencesProfessor of OphthalmologyHarvard Medical SchoolHarvard [email protected]

Jacque Duncan, MDProfessor, Clinical OphthalmologyDepartment of OphthalmologyUniversity of California, San [email protected]

David M. Gamm, MD, PhD (Co-Chair)Associate Professor and DirectorDepartment of Ophthalmology and Visual SciencesMcPherson Eye Research InstituteUniverity of [email protected]

Wei Li, PhDSenior Investigator

Retinal Neurobiology SectionNational Eye InstituteNational Institutes of [email protected]

Pamela Raymond, PhDStephen S. Easter Collegiate ProfessorDepartment of Molecular, Cellular, andDevelopmental BiologyCollege of Literature, Science, and the ArtsUniversity of [email protected]

Thomas Reh, PhDProfessorDepartment of Biological StructureUniversity of Washington School of [email protected]

Austin Roorda, PhDProfessorVision ScienceSchool of OptometryUniversity of California, [email protected]

Joshua R. Sanes, PhDProfessor, Molecular and Cellular BiologyDirector, Center for Brain ScienceDepartment of Molecular and Cellular BiologyHarvard [email protected]

Paul Sieving, MD, PhDDirectorNational Eye InstituteNational Institutes of [email protected]

Jane C. Sowden, PhDProfessorUCL Institute of Child Health, Stem Cells andRegenerative MedicineUniversity College London, [email protected]

Masayo Takahashi, MD, PhDProject LeaderLaboratory for Retinal RegenerationCenter for Developmental [email protected]


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Sally TempleScientific DirectorNeural Stem Cell [email protected]

Budd A. Tucker, PhDAssistant ProfessorOphthalmology & Visual SciencesUniversity of Iowa Carver College of [email protected]

Monica Vetter, PhDProfessor and ChairDepartment of Neurobiology and AnatomyUniversity of [email protected]

Shu-Zhen Wang, PhDProfessor of OphthalmologyUniversity of Alabama at [email protected]

Rong Wen, MD, PhDProfessor of OphthalmologyBascom Palmer Eye [email protected]

David R. Williams, PhDDean for Research in Arts, Science, and EngineeringCenter for Visual ScienceUniversity of [email protected]

Rachel Wong, PhD (Co-Chair)ProfessorDepartment of Biological StructureUniversity of [email protected]

Wai T. Wong, MD, PhDInvestigatorUnit on Neuron-Glia Interactions in Retinal DiseaseNational Eye InstituteNational Institutes of [email protected]

Ting Xie, PhDInvestigator and professorDepartment of Cell Biology and AnatomyUniversity of Kansas Medical CenterDepartment of OphthalmologyUniversity of Missouri School of Medicine at KansasCityStowers Institute for Medical [email protected]

Kang Zhang, MD, PhDProfessor or Ophthalmology and Human GeneticsDirectorInstitute for Genomic MedicineShiley Eye InstituteDepartment of OphthalmologyUniversity of California, San [email protected]


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