Gene Therapy Using Stem Cells - CSHL...

Gene Therapy Using Stem Cells

Erin R. Burnight1, Luke A. Wiley1, Robert F. Mullins1, Edwin M. Stone1,2, and Budd A. Tucker1

1The Stephen A. Wynn Institute for Vision Research, Department of Ophthalmology and Visual Sciences,University of Iowa, Iowa City, Iowa 52242

2Howard Hughes Medical Institute, University of Iowa, Iowa City, Iowa 52242

Correspondence: [email protected]

Viral-mediated gene augmentation therapy has recently shown success in restoring vision topatients with retinal degenerative disorders. Key to this success was the availability of animalmodels that accurately presented the human phenotype to test preclinical efficacyand safety.These exciting studies support the use of gene therapy in the treatment of devastating retinaldegenerative diseases. In some cases, however, in vivo gene therapy for retinal degenerationwould not be effective because the cell types targeted are no longer present. The develop-ment of somatic cell reprogramming methods provides an attractive source of autologouscells for transplantation and treatment of retinal degenerative disease. This article exploresthe development of gene therapy and patient-derived stem cells for the purpose of restoringvision to individuals suffering from inherited retinal degenerations.

Gene therapy for the treatment of inheriteddisorders such as adrenoleukodystrophy

(Cartier et al. 2009), adenosine deaminase-relat-ed severe combined immunodeficiency (Gasparet al. 2011), and the childhood blindness Lebercongenital amaurosis (LCA) (Bainbridge et al.2008; Hauswirth et al. 2008; Maguire et al. 2009)have recently shown great promise. Inherent tothe success of these clinical trials was the avail-ability of animal models to test therapeutic strat-egies. For instance, gene replacement with anadeno-associated virus (AAV) vector to treatRPE65-associated LCA (LCA2) was largely suc-cessful in both the Rpe652/2 mouse andthe large Briard dog model of the disease (Ben-nicelli et al. 2008). In comparison to con-tralateral sham-injected eyes, electroretinogram

(ERG) responses and visual acuity increased sig-nificantly in Rpe652/2 mouse eyes treated sub-retinally with an AAV-RPE65 vector. Similarly,subretinal AAV-RPE65 delivery resulted in sig-nificant reversal of nystagmus and visual deficitsin RPE65 mutant dogs (Acland et al. 2001, 2005;Narfstrom et al. 2003). Notably, ERG a-waves,indicative of photoreceptor responses, also in-creased dramatically (by 26%–60%) and per-sisted for the duration of the study followinginjection of the therapeutic gene transfer vector.The kinetics of the a-waves were not significantlydifferent between treated and phenotypicallynormal animals (Bennicelli et al. 2008). Thesepromising findings paved the way for the devel-opment of AAV-mediated RPE65 gene replace-ment therapy for patients with LCA2.

Editors: Eric A. Pierce, Richard H. Masland, and Joan W. Miller

Additional Perspectives on Retinal Disorders: Genetic Approaches to Diagnosis and Treatment available at

www.perspectivesinmedicine.org

Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a017434

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ANIMAL MODELS AND THERAPEUTICGENE TRANSFER

Although animal models are invaluable for in-vestigation of disease pathophysiology and eval-uation of gene transfer strategies, not all modelsfaithfully recapitulate the human phenotype.This is especially true when developing thera-peutic strategies for the treatment of inheritedmacular degenerations. For example, the studyof gene transfer for the treatment of age-relatedmacular degeneration (AMD), Stargardt dis-ease, and Best disease are hampered by the factthat the rod-dominant mouse retina lacks amacula, the central zone of the primate retinathat has evolved to subserve very fine visual acu-ity. Similarly, many disease models have a dras-tically different age of onset and disease progres-sion than is observed in the human condition.For instance, dominant Pro23His rhodopsin-associated retinitis pigmentosa typically causesrelatively late-onset disease in humans, with sig-nificant vision remaining until late in life. Thepig model of Pro23His rhodopsin-associatedretinitis pigmentosa has a retinal disease thatis significantly more aggressive than that seenin humans; as early as 3 mo of age, these ani-mals have extensive photoreceptor cell loss (Fig.1) (Ross et al. 2012). Similarly, in the rd16CEP290 mouse model of LCA, mice are bornwith a full complement of photoreceptor cells,but the very aggressive disease results in almostcomplete loss of photoreceptor cells by 3 wk ofage (Chang et al. 2006). The fact that a largeproportion of the photoreceptor cells are lostbefore the eyes open makes gene transfer ap-proaches for the treatment of this disease verydifficult.

Advances in pluripotent stem cell technolo-gies provide an exciting avenue for the treatmentof retinal degenerative disease. For instance, hu-man embryonic stem cell (ESC)-derived photo-receptor precursor cells transplanted into amouse model of retinal degeneration havebeen shown to migrate into and integrate withinthe degenerative retina, express rod and conephotoreceptor markers and restore electroreti-nal activity (Lamba et al. 2009). However, ESC-derived photoreceptor transplants are by defini-

tion allogeneic and would likely require life-longimmune modulation if used clinically (Preynat-Seauve et al. 2009; West et al. 2010). Like ESCs,patient-specific induced pluripotent stem cells(iPSCs) possess the ability to differentiate intophotoreceptor precursors which can integrateinto a diseased retina and give rise to new func-tional photoreceptor cells (Lamba et al. 2010;

Figure 1. The transgenic pig carrying a humanPro23His rhodopsin transgene displays drastically ac-celerated retinal degeneration. A–B: Histologicalanalysis of both wild-type (A) and Pro23His (B) pigretinal tissue at 6 mo of age. C–D: Cytochemicalanalysis of both wild-type (C) and Pro23His (D)pig retinal tissue at 6 mo of age, using wheat germagglutinin (WGA, red) and DAPI (blue).

E.R. Burnight et al.

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Homma et al. 2013; Tucker et al. 2013b). How-ever, unlike ESCs, iPSCs can be autologous andthus have a greatly reduced risk of being rejectedby the host immune system.

Patient-derived iPSCs can be generated in4–6 wk via forced factor expression in a varietyof cell types. Takahashi and Yamanaka firstshowed this concept through retroviral-mediat-ed expression of the transcription factors OCT4,SOX2, KLF4, and cMYC in mouse embryonicfibroblasts (Takahashi and Yamanaka 2006)and human primary dermal fibroblasts (Taka-hashi et al. 2007). In addition to the “Yamanakafactors,” Yu and colleagues in the Thomson lab-oratory established that the factors OCT4,SOX2, LIN28, and NANOG were capable of gen-erating induced pluripotent stem cells frommesenchymal cells (Yu et al. 2007). Many celltypes have since been reprogrammed usingforced factor expression including dermal kera-tinocytes (Carey et al. 2009), neural progenitorcells (Kim et al. 2011), hepatocytes (Liu et al.2010), and peripheral blood cells (Loh et al.2009). Since Yamanaka’s seminal report, severalgroups showed reprogramming using many de-livery methods—both integrating and noninte-grating. Integrating lentiviral vectors are widelyused for delivery of reprogramming transgenes(Brambrink et al. 2008; Maherali et al. 2008).However, to avoid the insertional mutagenicrisk associated with integrating vector systems,investigators use adenovirus (Stadtfeld et al.2008), Sendai virus (Jin et al. 2012; Tuckeret al. 2013a), and transient plasmid delivery(Gonzalez et al. 2009; Si-Tayeb et al. 2010). Fora more detailed description of these and otherdelivery methods please refer to reviews (Malikand Rao 2013; Hu 2014).

Beyond being an attractive cell source forretinal transplantation, the patient-specific na-ture of the iPSC makes this technology veryattractive for disease modeling and investiga-tion of disease pathophysiology. That is, themolecular causes of clinical variations in diseasecan be studied by comparing cells derived frommildly and severely affected individuals. For ex-ample, fibroblast cultures from CEP290-associ-ated LCA patients with different genotypesshow cellular phenotypes of varying degrees of

severity (Burnight et al. 2014). iPSC-derivedphotoreceptors from each of these patients canbe used to study the molecular causes of thephenotypic variation observed. The efficacy ofnovel therapies can also be tested on cells of theactual patients who might be treated.

INDUCED PLURIPOTENT STEM CELLSAS PATIENT-SPECIFIC MODELS TO TESTEXOGENOUS GENE DELIVERY

There are many vector systems from which tochoose when developing a strategy for the treat-ment of an inherited retinal degenerative dis-ease. For in vivo gene transfer, there are severalobstacles to the effective delivery and expressionof therapeutic transgenes. In many cases, genedelivery methods must overcome host immuni-ty (Reichel et al. 1998; Ikeda et al. 2002; Li et al.2008). Moreover, certain cell and tissue typesare difficult to transduce in vivo, which is likelybecause of a combination of incompatible vec-tor tropism and limited diffusion away from theinjection site. Gene delivery to cells ex vivo (i.e.,“in a dish”), circumvents many of these limita-tions and shifts the scientific focus from thedelivery vehicle to the genetic cargo. This is ofparticular value when one wishes to chooseamong several possible promoters, evaluategene function, and determine the optimal levelof transgene expression without killing dozens,if not hundreds, of animals in the process.

One of the major considerations when de-termining which vector system to use is genesize. Small, nonstructural enzymes (i.e., MAK,RPE65, etc.) are often well-suited for expressionvia the AAV vector system (Bennicelli et al. 2008;Conlon et al. 2013; Molday et al. 2013; Dai et al.2014). Indeed, the recent success of clinical tri-als involving patients with LCA2 highlights theutility of the AAV vector for in vivo gene transferto the retina (Bainbridge et al. 2008; Hauswirthet al. 2008; Maguire et al. 2008). However, thenatural serotypes of AAV do not transduce hu-man pluripotent stem cells very efficiently. Thecommonly used AAV2 serotype transduces hu-man stem cells only marginally (10% at a mul-tiplicity of infection of 10,000 [Asuri et al.2012]). Asuri and colleagues (2012) addressed


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this limitation via directed evolution of the cap-sid, which yielded threefold higher transduc-tion efficiency when transduced at an equalmultiplicity of infection. Another considerationwhen using AAV vectors in gene transfer studiesis the packaging limit. Large genes such asCEP290 (�8 kb coding sequence) or ABCA4(�6.5 kb coding sequence) require vectors thatcan accommodate more than the 4.5 kb packag-ing limit of AAV (Wu et al. 2010). To this end, thelentivirus is an attractive choice. The packaginglimit of lentivirus is �8210 kb (Balaggan andAli 2012) and can thus accommodate largertransgene expression cassettes. Moreover, len-tiviruses can be pseudotyped with a variety ofpantropic envelope glycoproteins (Calameet al. 2011)—the most common of which is theG glycoprotein of the vesicular stomatitis virus(VSVG)—and can transduce pluripotent stemcells well (Endo et al. 2007; Tucker et al. 2011).

We have employed lentiviral-mediated genetransfer to transduce pluripotent stem cell-de-rived photoreceptor precursor cultures frompatients with LCA and retinitis pigmentosa.Forexample, the most common cause of retinitispigmentosa in the Jewish population is a splice-altering insertion of an Alu element in the malegerm cell-associated kinase (MAK) gene. Ciliaformation assays showed that patient’s cells gen-erated elongated cilia when compared with phe-notypically normal cells (Tucker et al. 2011). Ontransductionwith VSVG-pseudotyped lentiviralvectors expressing MAK, cells formed signifi-cantlyshorterciliacompared with untransducedcultures. Thus, we achieved functional genecorrection in patient-specific, stem cell-derivedphotoreceptor precursors. For treatment of pa-tients with LCA, we successfully packaged cDNAencoding the large structural gene CEP290 intolentiviral vectors with the heterologous cyto-megalovirus (CMV) promoter driving trans-gene expression (Burnight et al. 2014).

Another important consideration in devel-oping gene transfer strategies to treat inheriteddisorders in vitro is the timing of the geneticcorrection. Is it more effective to perform genetransfer to the somatic cells (i.e., fibroblasts orkeratinocytes) before reprogramming them intoiPSC cultures, or to differentiate the cultures

first and then transduce them? Long-term geneexpression in immature cell types transducedwith g-retroviral and lentiviral vectors can besilenced because of epigenetic mechanismssuch as DNA methylation or histone modifica-tion of the heterologous promoter (Ellis 2005;Herbst et al. 2012). For example, transgenesilencing was observed in iPSCs derived from amurine model of tyrosinemia type 1 (FAH – / –)after transduction with a lentivirus expressingFAH (Wu et al. 2011). Only 20% of cells ex-pressed the transgene. Nonetheless, a smallpercentage of these genetically corrected iPSCsshowed full pluripotent potential, and viablemice were generated via tetracomplementation(Wu et al. 2011).

Because phenotypic correction is not as ef-ficient when transducing iPSCs (because oftransgene silencing), it may be more advanta-geous to correct somatic cells before reprogram-ming. For example, Raya and colleagues trans-duced fibroblasts from Fanconi anemia patientswith lentiviral vectors expressing FANCA orFANCD2. These cells, on reprogramming, ex-pressed the pluripotency markers OCT4,SOX2, NANOG, SSEA3, SSEA4, and TRA1-60,and were indistinguishable from human embry-onic stem cells and iPSCs from healthy individ-uals. Moreover, the genetically corrected iPSCsdifferentiated into phenotypically normal mye-loid and erythroid progenitors (Raya et al. 2009).

Introducing therapeutic transgenes after dif-ferentiation is also a viable option in some cases.We recently showed successful lentiviral genetransfer to differentiated photoreceptor precur-sor cultures from patients with CEP290-associ-ated LCA. Transduced photoreceptor precursorcells (identified by expression of retinal mark-ers OTX2, OPSN1SW, and ROM1) expressedCEP290 transcript and full-length protein after90 d of differentiation (Burnight et al. 2014).Furthermore, a cilia formation defect that wasidentified in serum-starved fibroblasts isolatedfrom a CEP290 patient carrying two hypomor-phic alleles was rescued following transductionwith the CEP290-expressing lentiviral vector de-scribed above (Burnight et al. 2014).

Effective gene therapy strategies must resultin therapeutic transgene expression in the ap-



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propriate cell type and at the appropriate level.Transgene expression is commonly driven bystrong, ubiquitously expressing promoter ele-ments, such as CMV. However, overexpressionof large structural proteins such as CEP290 ormembers of the BBSome, a complex of proteinsassociated with Bardet-Biedl syndrome, maylead to cytotoxicity and cell death (Seo et al.2013; Burnight et al. 2014). For an inheritedretinal disease, it is important to direct the ap-propriate level of expression in the cell type spe-cific to the pathophysiology of the disease. Thus,developing a “molecular toolbox” of gene trans-fer reagents is of great interest. This array ofreagents would include promoters specific toheterologous cell or tissue types with varyingdegrees of expressivity and a series of viral vec-tors with a range of carrying capacities (Fig. 2).Indeed, work from our laboratory and othersindicates promoter elements from retina-specif-ic genes such as GRK1, IRBP, RHO, PDE, NRL,and RCVRN achieve photoreceptor-specific ex-pression both in vitro and in vivo (Nicoud et al.2007). However, it has yet to be shown whether

issues relating to the level of transgene expres-sion and cellular toxicity will remain. For somegenes, precise stoichiometry may require iden-tification and use of the endogenous promoterof the therapeutic gene. Conversely, a series ofcell type-specific promoters, such as those indi-cated above, with tunable expression elementsmay be useful. The ability to efficiently generateand test these reagents will almost certainly ben-efit from the ability to create disease-specific celltypes from affected individuals.

GENOME EDITING

As indicated above, successful genetic correc-tion in iPSCs was achieved through gene aug-mentation using exogenous vector systems.Therapeutic transgene expression in the retinapersists for long periods of time, reducing theneed for vector readministration (Bemelmanset al. 2008; Ikeda et al. 2009; Koirala et al.2013; Testa et al. 2013). However, integratingvectors such as those derived from g retroviruscarry the inherent risk of inducing insertional

Constitutive promoter

Expression level

EF1a

Vec

tor

HSV Rhok-Ush2A

Car

go s

ize

AAV

Lenti

PhotoreceptorRhoK, Rec, GNAT2

Cell type specific promoter

RPERPE65, BEST1, MITF

HSV

CMV

RhoK

Ush2A

Figure 2. Schematic diagram depicting a molecular toolbox for exogenous replacement of genes. Genes ofvarious sizes requiring various levels of expression and cell type selectivity.



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mutagenesis (Hacein-Bey-Abina et al. 2003). Aswas shown by Hacein-Bey-Abina et al., somepatients who received ex vivo gene augmenta-tion via a g-retroviral vector developed a T cell-like leukemia as a result of vector insertion andproto-oncogene activation in treated cells. Thisadverse event highlights the mutagenic risk in-volved with employing integrating vectors. Fur-thermore, given the fact that precise regulationof gene expression may be required, the abilityto genetically modify defective genes in vitroprovides an attractive strategy for both the cre-ation and the repair of disease-causing muta-tions. Development of patient-specific autolo-gous cells for transplantation would greatlybenefit from such a strategy.

The use of genome editing tools such as zincfinger nucleases (ZFNs), transcription activa-tor-like effector nucleases (TALENs), and therecently reported clustered regularly inter-spersed short tandem repeat (CRISPR)/Cassystem is increasing. By editing the mutationdirectly through double-strand break inductionby a genome editing reagent and subsequent ho-mology-directed repair, one can take advantageof the endogenous expression control elements(i.e., promoters, enhancers, and repressors).This approach addresses some of the more seri-ous limitations of gene replacement such as in-appropriate spatial expression or incorrect levelsof expression from heterologous promotersas well as the possibility of activation or inacti-vation of deleterious genes from vector inte-gration.

In nature, transcription activator-like (TAL)effector proteins are transcription factors usedby the plant pathogen Xanthamonas to evadehost immune mechanisms (Gu et al. 2005; Kayet al. 2007; Sugio et al. 2007). The DNA target-ing domain in the TAL effector protein consistsof tandem repeats of �34 amino acids, at thecenter of which is a highly variable pair of aminoacids that confer nucleotide specificity (Schor-nack et al. 2006). The DNA recognition “code”was recently deciphered (Boch et al. 2009; Mos-cou and Bogdanove 2009), allowing TAL effec-tors to be engineered to recognize specific sitesin the genome. Complexing the engineered TALeffectors with the nuclease domain of the type-

II restriction endonuclease FokI can inducedouble-strand breaks at specific sites at or nearmutations in patient-specific iPSCs. When co-delivered with a donor cassette containing a cor-rected sequence, homology-directed repair canresult in genetic correction of these cells.

Recently Hockemeyer and colleagues (2011)targeted three endogenous loci, PPP1RP12C,OCT4, and PITX3, with engineered TALENs.TALEN expression constructs and donor cas-settes were electroporated into both humanESCs and iPSCs. The researchers reported tar-geting efficiencies of 50%–100% depending onthe locus. To assess binding specificity of thePPP1RP12C TALEN pair, Hockmeyer et al. in-terrogated the 19 top-ranked, maximal-likeli-hood potential off-target sites, as determinedusing systematic evolution of ligands by expo-nential enrichment (SELEX)-derived base-fre-quency matrices (Perez et al. 2008). NextGenIllumina sequencing revealed insertions or de-letions at off-target sites at a very low rate(0.01%), indicating the TALENs were quite spe-cific. Southern analysis at all three sites indicat-ed modification at the intended site only, con-firming these results (Hockemeyer et al. 2011).This valuable series of experiments providessupport for the feasibility of successful genomemodification in pluripotent stem cells, and sug-gests a powerful method for employing geneticrepair in patient-specific, iPSC-derived photo-receptor precursor cells for treatment of inher-ited retinal degenerations. That said, beforeclinical use, it will be important to assess off-targeting in cells treated with genome editingreagents to prevent unwanted outcomes. Dou-ble-strand breaks are induced via heterodimeri-zation of the FokI nuclease domains from twoengineered TALENs targeted to half-recognitionsequences at the intended locus. An unintendedoutcome of TALEN expression is the formationof homodimers, from which off-target cleavagecan result. Thus, reengineering the FokI catalyt-ic domain such that TALEN pairs form obligateheterodimers reduces the frequency of off-targetcleavage (Doyon et al. 2011; Hockemeyer et al.2011; Ramalingam et al. 2011).

Recently, the CRISPR/Cas system has beenintroduced (Cong et al. 2013; Jinek et al. 2013;



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Mali et al. 2013) and offers several advantagesfor genome editing in iPSCs. First, CRISPR re-agents are easier to design and work more pre-dictably because their DNA targeting is basedon RNA hybridization instead of the zinc-fingerprotein binding of TALENs. The guide RNA ofthe CRISPR system consists of an NGG se-quence (protospacer adjacent motif, or PAM)downstream from the 20-nucleotide target(Cho et al. 2013; Cong et al. 2013; Mali et al.2013). One can generate a CRISPR targetingreagent by simply synthesizing these guide se-quences as complementary oligonucleotidesand ligating them into a bicistronic vector thatalso contains a Cas9 nuclease optimized for ex-pression in human cells (Cong et al. 2013; Ranet al. 2013b).

Recently, Mali and colleagues (2013) showedsuccessful genome editing in human iPSCs us-ing the CRISPR/Cas system. These researchersdeveloped humanized CRISPR/Cas reagentstargeting the endogenous AAVS1 locus. Theyshowed homology-directed repair using a dou-ble-stranded DNA donor cassette in 2%–4%of fibroblast-derived iPSCs generated fromparticipants in the Personal Genome Project(PGP).

As with TALEN and ZFN technology, thepossibility of toxicity from off-target double-stranded breaks must also be addressed whenemploying the CRISPR/Cas system (Fu et al.2013). Targeting via CRISPR/Cas can toleratemispairing of the 20-nucleotide guide RNA(Cong et al. 2013; Jinek et al. 2013), which cre-ates the potential for double-stranded breaks atunintended genomic loci. To address this issue,Cong et al. (2013) mutated a single amino acidin the catalytic domain of the Cas9 nuclease,thereby limiting the enzyme’s activity to sin-gle-strand “nicks.” These “nicks” can be re-paired by the endogenous base excision repairpathway (Lindahl 1993). By using guide RNAswith a mutant Cas9 “nickase,” targeted to se-quences on opposite strands, one can increaseediting specificity and significantly reduce off-target toxicity. For example, Ran and colleagues(2013a) recently showed efficient modifica-tion (.40%) at three loci in human cells usingpaired nickases targeting sequences separated

by four to 20 bases on opposite strands of ge-nomic DNA. Moreover, using deep sequencinganalysis of off targets, these investigatorsshowed a 200- to 1500-fold increase in specific-ity when compared with wild-type Cas9. Ranand colleagues extended these studies in vivoby coinjecting guide RNAs targeting theMecp2 locus and either mutant or wild-typeCas9 mRNA into single-cell mouse zygotes.These experiments yielded .80% modifica-tion at the intended locus. A previous studyalso supported in vivo genome editing withthe CRISPR/Cas system. Wang et al. injectedwild-type Cas9 and guide RNAs targeting theTet1 and Tet2 loci as well as oligo repair tem-plates into zygotes and generated mice withmodification of both genes at a rate of 80%(Wang et al. 2013). This elegant set of experi-ments show the utility of employing nickases toincrease specificity and safety of the valuablegenome editing CRISPR/Cas technology forboth in vitro and in vivo studies (Ran et al.2013a; Wang et al. 2013). To treat patientswho have molecularly confirmed retinal degen-erative disorders, CRISPR/Cas reagents can bedelivered to patient-derived iPSCs to correctdisease-causing mutations before differentiat-ing into photoreceptor precursor cells for trans-plantation. For in vivo therapeutic use, the ad-dition of inducible promoters to allow transientexpression of the guide RNAs may be desirableto further reduce off-target effects. ShouldCRISPR/Cas technology prove safe and effec-tive for in vivo studies, many of the challengesassociated with delivering large transgenes andregulating their level of expression may be over-come.

CONCLUDING REMARKS

In the not-too-distant past, the prognosis forpatients affected with an inherited retinal disor-der was grim. Patients were often told that therewas nothing that could be performed and thattheir fate was irreversible blindness. Fortunately,with developments in stem cell and gene trans-fer technologies there is now realistic hope fortreating many of these individuals. The abilityto accurately identify a patient’s disease-causing



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mutations is a prerequisite to the developmentof both gene-based and autologous cell-basedtherapies. As depicted in Figure 3, one can en-vision a strategy in which high-quality genetictesting, coupled with in vitro patient-specific,stem cell-based model systems, will allow forthe rapid development of gene- and cell-basedtreatments. This process would begin by collect-ing blood and skin samples from the affectedpatient. DNA isolated from blood would besubjected to genetic screening to identify po-tential disease-causing mutations, and skin-derived fibroblasts or keratinocytes would beharvested for generation of iPSCs. To confirmpathogenic mutations and elucidate pathophys-iologic mechanisms, iPSC-derived photorecep-tor precursor cells, generated via directed differ-

entiation, could be subjected to a variety ofpathway-specific analyses. Disease-specific phe-notypes identified in these studies would beused to test and confirm the efficacy of genetransfer and genome editing approaches. Genetransfer vectors shown to be effective at correct-ing the disease phenotype would then be used totreat patients’ remaining photoreceptor cells.For patients with extensive photoreceptor cellloss, an autologous cell replacement strategyusing patient-specific photoreceptor precur-sor cells genetically corrected via CRISPR- orTALEN-based genome editing would also beemployed. This approach could be used to treatinherited retinal disorders, regardless of theclinical stage and prevalence of the disease,and regardless of the size of the causative gene.

Sample collection

Blood

Sequencing

Genomic correction ofpatient specific cells

Test efficacy ofgene therapeutics

AAVLenti

HSV

Interrogate pathophysiology

Gene- and cell-based therapies

iPSCgeneration

Skin

Figure 3. Schematic diagram depicting the proposed pipeline from gene discovery to treatment of inheritedretinal degenerative disease.



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ACKNOWLEDGMENTS

This work was supported by National Institutesof Health (NIH) Directors New InnovatorAward 1-DP2-OD007483-01, NEI EY017451,F32 EY022834, Howard Hughes Medical Insti-tute (HHMI), Foundation Fighting Blindness,Stephen A. Wynn Foundation, Grousbeck Fam-ily Foundation, and Leo, Jacques & MarionHauser Family Vision Restoration Fund.

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November 13, 20142015; doi: 10.1101/cshperspect.a017434 originally published onlineCold Spring Harb Perspect Med

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http://perspectivesinmedicine.cshlp.org/cgi/collection/ For additional articles in this collection, see

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