Convergence of Human Genetics and AnimalStudies: Gene Therapy for X-Linked Retinoschisis

Ronald A. Bush1, Lisa L. Wei2, and Paul A. Sieving2

1National Institute on Deafness and Other Communication Disorders, Bethesda, Maryland 208922National Eye Institute, Bethesda, Maryland 20892

Correspondence: bushr@nidcd.nih.gov

Retinoschisis is an X-linked recessive genetic disease that leads to vision loss in males. X-linked retinoschisis (XLRS) typically affects young males; however, progressive vision losscontinues throughout life. Although discovered in 1898 by Haas in two brothers, the under-lying biology leading to blindness has become apparent only in the last 15 years with theadvancement of human genetic analyses, generation of XLRS animal models, and the devel-opment of ocular monitoring methods such as the electroretinogram and optical coherencetomography. It is now recognized that retinoschisis results from cyst formations within theretinal layers that interrupt normal visual neurosignaling and compromise structural integrity.Mutations in the human retinoschisin gene have been correlated with disease severity of thehuman XLRS phenotype. Introduction of a normal human retinoschisin cDNA into retino-schisin knockout mice restores retinal structure and improves neural function, providingproof-of-concept that gene replacement therapy is a plausible treatment for XLRS.

X-linked retinoschisis (XLRS) is a develop-mental retinal genetic disease that impairs

vision, typically beginning in young males andprogressing into older age. Although the con-dition was first recognized in the 19th centuryand is readily diagnosed clinically by the char-acteristic intraretinal cysts arranged in a “spoke-wheel” pattern in the center of the macula,scientific progress lagged until the gene wasidentified about 15 years ago. This led to under-standing the molecular and cellular basis of thecondition, discerning the structure and func-tion of the retinoschisin protein, generatingXLRS animal models, developing suitable deliv-ery vectors for the back of the eye, and methods

to monitor structure and function of the retinain animals and humans. The culmination ofthese scientific discoveries poises the field fornear-term clinical testing of a genetic therapydesigned to restore vision and prevent furthervision loss in XLRS patients.

CLINICAL DISEASE

Human retinoschisis leads to vision loss thataffects boys, typically at a young age. The diseasewas first described in two brothers in Germanyin 1898 (Haas 1898). The term retinoschisis wasfirst used to describe the condition by Wilczek(1935) and refers to the abnormal splitting

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

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Copyright # 2015 Cold Spring Harbor Laboratory Press; all rights reserved; doi: 10.1101/cshperspect.a017368

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through the retinal neurosensory layers. Retino-schisis is quite different from retinal detach-ment, in which the neurosensory tissue is ele-vated off and detached from the underlyingretinal pigment epithelium. In 1938, the diseasewas recognized as an X-linked recessive trait(Mann and MacRae 1938). Today, clinical prev-alence of retinoschisis is approximately 1:5000to 1:25,000 males worldwide (George et al.1995). Vision impairment is characteristicallynoticed in young males before grade school,although on occasion infants may manifest se-vere disease. Being an X-linked macular dystro-phy, the trait is passed from mothers to affectedmales. Female carriers nearly never displayretinal abnormalities and have normal electro-retinogram (ERG) responses. On very rare oc-casions, female carriers will show white flecks orareas of schisis within the peripheral retina (Ka-plan et al. 1991). However, because the vision offemale carriers is not impacted, this indicatesthat haploinsufficiency does not alter apparentretinal architecture nor result in obvious visualdeficiencies.

Individuals with congenital XLRS manifesta broad spectrum of clinical phenotypes, thusmaking initial diagnosis challenging. Schisiscan involve the superficial, middle, and deep

retinal layers, and is seen in both the nuclearand plexiform layers. Retinoschisis may involvethe peripheral retina (Prenner et al. 2006) andlead to increased incidence of full thicknessretinal detachment. In general, patients withX-linked juvenile retinoschisis show a mild re-duction in central visual acuity, which graduallydecreases, ranging from 20/60 to 20/120. Fig-ure 1 shows the hallmarks of the clinical signs inthe retina of a patient with a spokewheel patternof tiny cysts that reigns in the fovea (Fig. 1A).These cavities are also readily seen by opticalcoherence tomography (OCT) (Fig. 1B).

The ERG is one of the most clinically in-formative tests for X-linked retinoschisis. Thedark-adapted b-wave in XLRS patients is re-duced, whereas the a-wave amplitude often re-mains normal (Fig. 1C) (Peachey et al. 1987).This can result in an electronegative responsein which the positive-going b-wave does notincrease above baseline. As the a-wave originatesfrom photoreceptors, this indicates relativelynormal photoreceptor activity. The b-wave orig-inates from bipolar cells that lie proximal tothe photoreceptors, and the electronegative re-sponse implicates defective synaptic transmis-sion. Although an electronegative ERG responseis not exclusive to XLRS individuals, only a few

Normal XLRSDark-adaptedERG

Dim Flash

Bright Flash

b-wave

a-wave

100 µV

A

B

C

Figure 1. Fundus photo, OCT scan, and ERG typical of patients with XLRS. Color photo of macular regionin X-linked retinoschisis patient (A) showing the characteristic intraretinal cysts arranged in a “spokewheel”pattern and corresponding OCT scan (B). Dark-adapted ERGs from both scotopic dim and bright stimulusflashes for an XLRS patient shows a characteristic reduction in b-wave amplitude relative to the preserveda-wave (C).

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other retinal dystrophies result in this waveformconfiguration. Hence, finding this ERG type ina suspect male with a family history of retino-schisis is clinical confirmation of XLRS. How-ever, some affected males have a more normalERG, as was reported previously (Sieving et al.2000; Bowles et al. 2011; Vincent et al. 2013)

MANAGEMENT OF XLRS

Currently, there are no approved treatmentsfor the retinal degeneration or for the retinalschisis cavities. Various experimental treatmentoptions have been considered, including the useof carbonic anhydrase inhibitors (e.g., dorzola-mide and acetazolamide) with the presumedmechanism of action being the reduction of ex-cess intraretinal fluid in cystic spaces (Apushkinand Fishman 2006; Ghajarnia and Gorin 2007).In one study, seven of eight patients showed adecrease in foveal lesions in one eye as evaluatedby OCT, and six patients were reported to havea modest improvement in visual acuity (Apush-kin and Fishman 2006). These findings have notbeen replicated and are not universally accepted.Treatment of a retinoschisin-deficient mousemodel of XLRS (see below) with dorzolamidefailed to produce short-term or long-term struc-tural improvement in the retina (Zhour et al.2012). Another proposed intervention is manip-ulation of the retina through surgical intraocu-lar vitrectomy that may reduce schistic cavities(Ikeda et al. 2008). Unfortunately, these ap-proaches do not address the fundamental path-ophysiology, and no claims have been made thatthey alter the clinical course.

GENE AND CELL BIOLOGY OF XLRS

Genetic mutations causing XLRS are in the Ret-inoschisin 1 (RS1) gene, which maps to Xp22and is composed of six exons (Sauer et al. 1997).Retinoschisin is a 224 amino acid secretory pro-tein that is present throughout the neural retina(Grayson et al. 2000; Molday et al. 2001; Reid etal. 2003; Takada et al. 2004). Retinoschisin geneexpression has been localized to photoreceptorsin both human (Grayson et al. 2000) and mouseretina (Reid et al. 1999; Grayson et al. 2000;

Molday et al. 2001; Takada et al. 2004), as wellas to neurons in the inner nuclear layer (Moldayet al. 2001; Takada et al. 2004), and ganglion celllayer (Takada et al. 2004) of mouse.

RS1 has a signal peptide sequence that al-lows for transport of the protein to the extracel-lular surface (Vijayasarathy et al. 2006, 2007). Italso contains a single discoidin domain (Saueret al. 1997; Grayson et al. 2000). Discoidin do-mains are associated with a family of extracellu-lar cell surface proteins that are involved in celladhesion and signaling and that interact withcollagens to regulate extracellular matrix mod-eling (Vogel et al. 2006). Ten cysteine residueswithin the retinoschisin discoidin domain areimportant for appropriate protein folding andthe formation and function of retinoschisin di-mers and octamers (Wu and Molday 2003). RS1is thought to be critical for cell adhesion duringthe normal development and maintenance ofretinal structure. This is consistent with otherdiscoidin domain-containing proteins and issupported by clinical observations in patientsand in mouse models.

Retinoschisin expression begins during ear-ly postnatal retinal development and is main-tained throughout adult life (Takada et al.2004). As Figure 2 shows, retinoschisin is pro-duced in the inner retina, namely, the ganglioncells, early in ocular development (top panel).However, as the eye develops, additional reti-nal neurons produce retinoschisin, includingthe outer retinal cells such as photoreceptors(bottom panel), as indicated by the presenceof retinoschisin on the inner segments of rods.Interestingly, RS1 protein is also associatedwith bipolar cells, suggesting that it may be crit-ical for maintaining the structural connection ofpostsynaptic and presynaptic elements in theouter plexiform layer that is necessary for thetransmission of a visual signal from photorecep-tors to bipolar cells (Molday et al. 2001; Reidet al. 2003; Takada et al. 2004). This implies thatmonitoring synaptic integrity by the ERG b-wave would be a suitable means for gaugingthis connectivity. These findings support thehypothesis that retinoschisin plays a criticalrole in maintaining cell–cell adhesion and inpreserving retinal structure and function.

Gene Therapy for XLRS

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HUMAN GENETICS FOR RS1 MUTATIONS

In XLRS patients, numerous RS1 mutations havebeen identified (The Retinal Consortium 1998;Eksandh et al. 2000; Simonelli et al. 2003).Mutations reported include missense changes,deletions, insertions, and splice site variants.Structure-function analyses reveal that the mostcommon variants, missense mutations, interfereby three primary means: (1) interrupting nor-mal secretion of retinoschisin; (2) disruptingproper octomerization; or (3) impacting nor-mal function of the protein. Interestingly, a largeproportion of these mutations are within thediscoidin domain (exons 4–6), suggesting thatthe discoidin domain is a critical componentfor RS1 function. Examination of the nature ofthe RS1 gene mutations predicts that �40% ofall the identified mutations represent null allelesand, therefore, would not produce function-al retinoschisin protein (Molday et al. 2012).Such mutations represent the human equivalentof genetic knockout models in mice (see below).The majority of cases found with missense mu-tations had minimal structure perturbations

and consequently, standard disease (Sergeev etal. 2010). However, removal or insertions ofcysteine residues or changes in the hydropho-bic core of retinoschisin resulted in maximumstructural perturbations that caused significantchanges in photopic b/a ratios (Sergeev et al.2013). Although difficult to show, in some cases,severity of disease correlates with genotype(Sergeev et al. 2010, 2013; Bowles et al. 2011).

X-LINKED RETINOSCHISIS MOUSEMODELS

Currently, three XLRS mouse models targetingthe murine ortholog of the human RS1 genehave been developed (Table 1). All are deficientin endogenous murine retinoschisin. One mod-el was engineered with the insertion of a LacZ/Neor cassette into exon 3, thus generating a nullmouse model. Zeng et al. (2004) inserted a neo-mycin gene into intron 1 that resulted in abla-tion of murine retinoschisin protein expression.The last mouse model system was producedusing N-ethyl-N-nitrosurea (ENU)-based ran-

1-d-old mouse – only GCs express RS protein (brown staining)

14-d-old mouse – all neurons express RS protein (brown staining)

MOUSE C57/BL6

GCs

GCs

RPE

Retinoschisin protein expression

BP

RPErods

P1

P14

Figure 2. Retinoschisin protein expression in the mouse eye begins immediately following neuronal develop-ment and is maintained during adult life. Early in development retinoschisin is produced in the retinal ganglioncells (P1, top panel); later, extensive retinoschisin expression is also observed in the photoreceptors, includingthe rod inner segments (P14, bottom panel). (From Takada et al. 2004; reprinted, with express permission fromthe authors.)

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dom mutagenesis that led to a single base-pairsubstitution in intron 2 (Jablonski et al. 2005).This created an alternate splice site producingtwo transcripts in male mice: a 10 bp insertionand a 26 bp deletion at the junction of exon 2 tointron 2. Within these two transcripts, prema-ture stop codons are formed, preventing syn-thesis of full-length retinoschisin protein. Allthree knockout models display structural andfunctional retinal phenotypes very similar tothat observed in humans with XLRS. ERG eval-uations in the three mouse models show thecharacteristic reduction of the dark-adapted b-wave as observed in XLRS patients. Histologi-cally, retina structure is disorganized, with mis-aligned cells and retinal splitting evident in theinner nuclear layer and structural abnormalitiesof synapses within the outer plexiform layer—hallmarks of the human disease. Figure 3 showsthe retinal structure and dampened ERGs in theRs12/Y mouse (Zeng et al. 2004).

GENE THERAPY FOR XLRS

Gene Delivery Considerations

The expression of retinoschisin (Rs1) in miceduring development and in adult tissue, along

with the pathology described in the XLRSmouse retina, suggests that retinoschisin is im-portant in normal development and mainte-nance of the retinal architecture, and this proteinappears to be critical in sustaining functionalsynaptic integrity (Takada et al. 2004; Kjell-strom et al. 2007). These findings support thestrategy of gene augmentation as a therapeu-tic approach for XLRS patients. Experimentalstudies introducing human RS1 in mouse mod-els of XLRS showed that a single administrationgave sustained expression beyond a year andrestored both structure and function of the ret-ina (Zeng et al. 2004; Min et al. 2005). Based onthese encouraging studies, efforts are now di-rected to develop a gene therapy approach forXLRS patients.

A first consideration involves selecting anappropriate means to deliver the gene. Thereare several methods that could be used to in-troduce the RS1 gene into the eye. Viral vectorshave typically shown the best efficiency with re-gard to delivering the gene of interest to retinalcells. Within the eye, several viral vectors haveadvanced or are advancing into clinical testing.Among these are vectors based on adeno-as-sociated virus (AAV), adenovirus, retrovirus,and lentivirus (Song et al. 2003; Chevez-Barrios

Table 1. Comparison of XLRS mouse models

Source Synonyms Target Category

Reported

abnormal

phenotypes

Bernhard H.F. Weber(Weber et al. 2002)

Rs1h2/Y Partial deletions ofexon 3 and 4 andintron 3 with a LacZ gene insertion

Targeted(reporter)

Nervous system,vision/eye

Paul A. Sieving(Zeng et al. 2004)

Rs12/Y Deletion of all of exon 1and partial deletionof intron 1 with aneomycin geneinsertion

Targeted(knockout)

Vision/eye

Tennessee Mouse GenomeConsortium (TMGC)—Monica Jablonski(distributed via TMGC,Northwestern University,and JAX Labs)(Jablonski et al. 2005)

44TNJRs1tmgc1/Rs1tmgc1

and Rs1tmgc/Yformerly called

Rs1htmgc1

T to C transition at thesecond base of intron2 of the Rs1h gene

Chemicallyinduce(ENU)

Nervous system,pigmentation,vision/eye

Gene Therapy for XLRS

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et al. 2005; Campochiaro et al. 2006; Bainbridgeet al. 2008; Hauswirth et al. 2008; Maguire et al.2008, 2009; Simonelli et al. 2010; Balaggan andAli 2012). Today, a large number of gene ther-apy constructs, in particular those for humanocular diseases, are utilizing AAV. AAV is a non-pathogenic dependovirus that replicates in thepresence of another active virus infection, oftenadenovirus or herpes simplex virus (Berns andGiraud 1996). AAV virions are nonenvelopedand typically encase single stranded DNA. Thereare at least 12 naturally occurring serotypesof AAV (designated AAV1–AAV12), althoughmore than 100 nonredundant forms have been

discovered (Mori et al. 2004; Daya and Berns2008; Vandenberghe and Auricchio 2012).

AAV has two open reading frames. The firstcontains rep, which encodes four proteins nec-essary for viral replication. The second com-prises cap, which encodes three structural pro-teins that make up the outer icosahedral capsidof the virus. The cap or capsid proteins havedifferent affinities for various cell surface re-ceptors. For instance, the primary receptors forAAV serotype 2 (AAV2) are heparin sulphateand aVb5 integrins (Summerford and Samulski1998; Summerford et al. 1999). AAV4 and AAV5preferentially bind sialic acid or platelet-derived

“Electro-negative ERG”

#551 #553

RGCIPL

INLOPL

ONL

ISOS

OPL schisis

INL schisis

WT eve Rs1–/y eye

1400

1200

1000

WT(n = 3)

Rs1– /y

(n = 3)

800

600

400

200

0–7 –6 –5 –4 –3

Intensity (log cd–s/m2)

Am

plitu

de (

µV)

–2 –1 0 1

Normal(#1276)

–6.4

–5.4

–4.4

–3.4

–2.4

–1.4

–0.4

–0.6

50 msec 50 msec

1000 µV

Stim

ulus

(lo

g cd

-s/m

2 )

500 µV

Rs1–/y (#1277)

Figure 3. Retinal structure and ERG function in the Rs1-KO (Rs12/y) mouse. The retinal sections show structuralchanges between the normal mouse eye and the RS-KO mouse eye. The Rs1-KO eye shows distortions in theretinal ganglion cell layer (RGC) and random cells in the inner plexiform layer (IPL). Likewise, the synapticconnections between photoreceptors and bipolar cells are irregular in the inner nuclear layer (INL) and outerplexiform layer (OPL). Structural gaps, equivalent to “retinoschisis cavities,” are evident in the INL. Photore-ceptors are reduced in the retinal sections in the KO mice versus normal mice, and some photoreceptor nucleilocalize to the inner and outer segment zones (IS and OS) rather than exclusively within the outer nuclear layer(ONL). As in XLRS humans, the Rs1-KO mouse ERG shows disproportionate b-wave reduction across the rangeof stimulus flash intensity, as is readily seen in the ERG wave form (arrows) and also in the graph of b-waveamplitude versus intensity. (From Zeng et al. 2004; reprinted, with express permission from the authors.)

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growth factor (Kaludov et al. 2002; Di Pasqualeet al. 2003), whereas AAV3, AAV8, and AAV9bind to laminin (Akache et al. 2006). Therefore,careful selection of viral serotype becomes animportant consideration in choosing an ap-propriate vector for gene delivery for a givendisease. This and the delivery route may be crit-ical for a successful gene-based interventionalapproach.

Choosing the correct viral vector can helpaddress another key component of a successfulgene therapy product, namely the appropri-ate expression of the replacement or restorativegene. Historically, transgene expression fromAAV vectors has a delayed onset, sometimes re-quiring up to 6 wk to achieve peak levels (Ben-nett et al. 1997). This would be detrimentalfor acute applications. However, two strategieshave been devised to overcome this limitation.The first is the development of modified or mu-tant capsids. Mutant capsids with specific ami-no acid substitutions have resulted in highertransgene expression in a shorter period oftime (Petrs-Silva et al. 2009). Investigators alsohave found that photoreceptors can be effective-ly transduced by intravitreal administration, afinding that has practical clinical implicationsfor both ophthalmologists and patients (see be-low) (Dalkara et al. 2011; MacLachlan et al. 2011;Petrs-Silva et al. 2011).

In addition to the gene delivery vector, thesecond consideration is the rate-limiting re-quirement for synthesis of a complementaryDNA strand from the original AAV single-strandDNA. Incorporating a second strand of DNAwithin the virus (self-complementary AAV) re-duces the time required for gene expression byremoving the necessity to synthesize a secondDNA strand. This also eliminates the need forhost DNA replication machinery. Using thisapproach it has been shown that transgene ex-pression can be measured within 4 d followingAAV-transgene introduction into the eye (Panget al. 2010).

A third component that is often critical forthe pharmacokinetic expression of the trans-gene is promoter selection. The cytomegalovi-rus (CMV) promoter is commonly used. Thispromoter can lead to high transgene expression

but in many systems the expression is transient,promiscuous and in all cell types. Because XLRSinvolves chronic degeneration of retinal cellsover years and decades in humans, an importantcriterion is selection of a promoter that leadsto prolonged gene production in multiple celltypes. Thus, for XLRS, several promoters in-cluding the rhodopsin (rho) and rhodopsin ki-nase (rho K) promoters have been investigated.Both of these promoters are specific to photo-receptors and give high expression, but they mayalso result in cell death, likely because of over-expression of retinoschisin (Y Zeng, unpubl.).Use of the retinoschisis promoter should fa-cilitate the expression of retinoschisin in all cellsin which it is normally found. By including theinterphotoreceptor retinoid binding proteinenhancer, gene expression levels following intra-vitreal injection reaches, in some animals, levelscomparable to that found in wild-type animals(see Fig. 4).

Routes of Administration

In the majority of ocular gene therapy trialsusing AAV, the genes are delivered via subretinalinjection (Bainbridge et al. 2008; Hauswirthet al. 2008; Maguire et al. 2008, 2009; Simonelliet al. 2010) with good success. This route ofadministration has the experimental advantageof direct dose scaling from the small mouse eyeto the larger human eye. However, this proce-dure is complex and requires pars plana vitrec-tomy surgery in an operating room, which has ahigh risk of complications (e.g., retinal tear orretinal detachment). XLRS patients are alreadyprone to retinal detachment, thus utilizing amethod that decreases this occurrence wouldbe preferred. Intravitreal injections into the eyehave become commonplace with the adventof Macugen, Lucentis, and Eylea (three agentsapproved for the treatment of wet age-relatedmacular degeneration), and can be repeatedmonthly. Moreover, intravitreal injections canbe administered in the office and given undertopical anesthesia, making this procedure lesscomplicated and more convenient for patient,physician, and clinical staff. However, there is

Gene Therapy for XLRS

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b-wave

Untreated: b-wave/a-wave = 1.3

Treated: b-wave/a-wave = 2.8

a-wave

ERG(function)

OCT(in vivo)

WT Rs1-KO

IHC(histology)

IPL

IPL

IPL

ONONON

IPL

IPL

IPL

IPL

IPLONL

ONL

ONLONL

ONL

cavities

ISONLIS

#7461–2.5e12 vg/eye

b-wave/a-wave= 2.0

250 mV

50 msec

Untreated (left eye) Treated (right eye)

Retinoschisin staining Cell nuclei

Figure 4. Retinal structure, retinoschisin expression and function in wild-type (WT), untreated- and treated-Rs1-KO mice. OCT in vivo images show the structure of a portion of the retinas centered on the optic nerve(ON) from a WTmouse and the right and left retinas of an Rs1-KO mouse treated in the right eye with 2.5 � 109

vg/eye of AAV8-retinoschisin vector. The darker bands are the nuclear layers of the retina, the thickest being theouter nuclear layer (ONL) containing the photoreceptor cells. The lighter bands above the ONL are the outerand inner plexiform (IPL) layers. In the untreated eye of the Rs1-KO mouse, these layers are much less dense andthe borders between the layers less distinct. There are large cavities spanning the region between the ONL andIPL, a region that contains the second order neurons, including bipolar cells and their synapses with photo-receptors. The right eye was treated at day P21 and evaluated 16 wk later. OCT shows the cavities have closed inthe treated retina, and the borders between the layers are more regular and distinct than in the untreated eyes.The layer margins are still less distinct than in the WT retina. Immunohistochemical (IHC) staining forretinoschisin protein (red) in the WT retina shows its presence in the plexiform layers, with particularly strongstaining in the inner segments (IS) of the photoreceptors. Specific staining for retinoschisin is completelylacking in the untreated eye of the Rs1-KO mouse but is very strong in the treated eye, indicating robustexpression of the therapeutic protein in the same cells as in WT. The faint staining below the ONL in theuntreated eye is nonspecific staining of the retinal pigment epithelium. Immunohistochemistry analyses showthat retinoschisin expression in photoreceptor inner segments in treated eyes are essentially comparable to WT.Image brightness intensity was adjusted against blue nuclear staining of DAPI. The ERG shows a-wave andb-wave responses to a bright flash of light, indicating phototransduction activity of photoreceptors and activityof the second order bipolar cells, respectively. Both are reduced in size in the Rs1-KO mouse retinas, but the ratioof the b-wave to the a-wave is pathologically reduced in the untreated eye while it is increased in the treatedretina. This is consistent with the structural findings indicating severe pathology in the retina postsynaptic tophotoreceptors in untreated retinas, with structure partially maintained or restored when treated by AAV8-retinoschisin. In most of our studies the vector is administered at day P21–P28, when the retina is young butfully mature. However, similar rescue of structure and function is observed when AAV8-retinoschisin is ad-ministered at 2 and 3 mo of age.

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not a clear method to scale from a small animaleye to the larger human eye.

Intravitreal administration of native AAVvectors has resulted in scattered and spottytransduction of ganglion cells at the surface ofthe retina, but the vectors do not penetrate toreach neurons deep in the rodent or monkeyeye (Dalkara et al. 2013). AAV2 variants withtyrosine mutations, or newer laboratory-cre-ated AAV vectors with capsid modifications,show the ability to penetrate deep into the reti-na (Petrs-Silva et al. 2009; Vandenberghe andAuricchio 2012; Dalkara et al. 2013). Conse-quently, intravitreal delivery of genes for retinaldegenerative diseases is becoming more prom-ising clinically. In the case of XLRS, data usingan AAV8 containing the RS1 gene (AAV8-reti-noschisin) delivered into the eyes of XLRS miceby intravitreal injection show robust RS1 stain-ing of neurons throughout the mouse retina,including photoreceptors, bipolar and ama-crine cells (Park et al. 2009). This appears spe-cific for the pathophysiology caused by XLRS, asthe same vector does not penetrate the wild-typemouse retina. Additionally, a clinical trial utiliz-ing AAV2 with soluble Flt for wet age-relatedmacular degeneration (NCT01024998) is usingintravitreal delivery, providing further supportthat intraocular injection is plausible for somevectors. The published preclinical studies usingAAV2 to deliver an antiangiogenic molecule inprimate eyes showed intraocular inflammationto the vector capsid, as the AAV2-null vectorgave similar findings (MacLachlan et al. 2011).Inflammation was transitory, initially appearingin some animals at 1 mo after intravitreal injec-tion, increasing over the next few months, andthen declining.

Transition to Efficacy Studies

Preclinical safety and efficacy studies in animalscan provide the foundation for human clinicaltrials and are helpful to establish a possible ben-efit to treatment that outweighs potential risks.As reviewed above, mice in which expression ofthe Rs1 gene has been disrupted resulting inretinoschisin knockout (Rs1-KO) mice are theonly animal models for XLRS. Structural and

functional studies of Rs1-KO mice have showna phenotype that closely mimics retinal pathol-ogy in XLRS (Zeng et al. 2004). Cavity forma-tion reaches a maximum by 4–6 mo, in parallelwith functional changes such as reduction inthe ERG b-wave to a-wave ratio (b/a ratio)(Kjellstrom et al. 2007). This change is compa-rable to XLRS in young adults when the ERGb-wave and b/a ratio tends to reach minimumin the most severe patients (Bowles et al. 2011).AAV8-retinoschisin efficacy studies in our lab-oratory using Rs1-KO mice are described below.Retinal function was measured using ERGs,and retinal structure was evaluated in vivowith OCT imaging. Both are common clinicalmeasures and are performed using identical orsimilar instruments to those used for humanclinical diagnosis. In addition, retinoschisin ex-pression was evaluated postmortem in treatedanimals using semiquantitative immunohis-tochemistry. This provides a combined assess-ment of therapeutic protein expression withboth structural and functional benefit followingvector administration.

Efficacy Study Results

Two groups of Rs1-KO mice received intravitrealinjections in one eye between 3 and 5 wk of ageof either an AAV8 vector carrying a self-compli-mentary copy of the human retinoschisin geneand promoter or vehicle alone. Injected doseswere 2.5 � 109 vector genomes (vg)/eye downto 1 � 106 vg/eye. ERGs were recorded between12 and 16 wk after injection, followed within2 wk by OCT imaging and removal of the eyesfor immunohistochemistry to evaluate retino-schisin expression. Figure 4 shows the resultsfrom the treated and untreated eyes of a sin-gle mouse injected with 2.5 � 109 vg/eye andevaluated 16 wk after injection compared witha wild-type mouse. The untreated eye of theRs1-KO mouse has large cavities spanning thelayers between the photoreceptor outer nuclearlayer and the inner plexiform layer. These inter-mediate layers are also thicker than in wild typeand have ill-defined boundaries, indicating adegree of disorganization compared with thewell-defined layers in the wild-type animal.

Gene Therapy for XLRS

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The cavities in the untreated retina appear asempty space in the histological sections, but invivo there are structural connections betweenthe inner and outer margins of the cavities sim-ilar to those in human XLRS (Fig. 1). The un-treated eye lacks retinoschisin expression, whichis apparent in the inner nuclear layer and outerplexiform layer and is strongest in the innersegments of the photoreceptors in the wild-type retina. The structure of the treated retinain vivo and in histological sections, however, issimilar to wild type in organization of the reti-nal layers and lack of cavities. Some disorgani-zation is still evident in the internal structureof the layers and the indistinct or irregular mar-gins of the layers. The treated retina shows heavyretinoschisin staining of inner segments andin the two plexiform layers between the rowsof retinal cell bodies. Retinoschisin expressionin this retina equals or exceeds that of wild type,although this is variable across injected eyes andsometimes within injected eyes.

The ERG of the wild-type mouse shows adark-adapted a-wave and b-wave in responseto a bright stimulus flash. The a-wave reflectsphototransduction in the rods, and the b-waveoriginates from bipolar cells postsynaptic to thephotoreceptors. Thus, a reduction of photore-

ceptor number or activity will produce a reduc-tion in both the a-wave and b-wave, becausepostsynaptic responses depend on photorecep-tor input. A reduction in the a-wave greater thanin the b-wave, that is, an increase in the b/aratio, is a hallmark of photoreceptor degenera-tions (Machida et al. 2000), because photore-ceptors greatly outnumber the bipolar cells,and this convergence tends to reduce the effectof photoreceptor loss vis-a-vis the b-wave gen-eration. The response from the untreated eye ofthe Rs1-KO mouse in Figure 4 shows a reductionin b/a ratio, indicating a disproportionate effecton the response of bipolar cells. This could bea result of structural and functional disruptionof bipolar cells themselves and/or the synapticsignaling between photoreceptors and thesesecond order neurons. The smaller a-wave com-pared with wild type indicates that there wassubstantial photoreceptor loss, which is also ap-parent as a reduction in the outer nuclear layer,as shown by OCT and histology. The increasedb/a wave ratio in treated eyes is consistent withthe relatively normal structural appearance ofthe retinal layers postsynaptic to the photo-receptors, coupled with a substantial loss ofphotoreceptors. Figure 5 shows dose–responseresults for Rs1-KO eyes injected with AAV8-ret-

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Figure 5. Dose response graphs for the ERG b-wave and b-wave to a-wave ratio in Rs1-KO mice treated withAAV8-retinoschisin. Rs1-KO mice were treated in one eye with different concentrations of vector or vehiclebetween 3 and 5 wk of age, and the ERG was recorded 12 to 16 wk after injection. The b-wave and the b-wave toa-wave ratio was significantly increased at doses of 1e8 and 2.5e9, relative to untreated eyes (multiple t-tests usingHolm-Sidak correction for multiple comparisons).

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inoschisin or vector controls. There was a sig-nificant improvement in the b-wave and b/awave ratio in eyes treated with 1 � 108 and 2.5� 109 vg/eye of AAV8-retinoschisin but notwith 1 � 107 vg/eye or vehicle. The size andnumber of cavities were significantly reducedat 1 � 108 and 2.5 � 109 vg/eye, and these ret-inas showed significant retinoschisin protein ex-pression, as assessed with immunohistochemis-try, compared with untreated eyes (Fig. 4)

CONCLUDING REMARKS

Based on the very promising mouse efficacystudies, the strong transduction of the retinausing AAV8 after intravitreous administration,and an acceptable safety profile in the Rs1-KOmouse, a next step is to advance to a phase Iclinical trial in XLRS patients. The data ob-tained to date from animal studies suggest sev-eral issues to be considered in designing earlyclinical trials of RS1 gene therapy. Each patientshould be followed carefully for any clinical in-dication of an ocular inflammatory responseto vector administration. Simultaneously, ther-apeutic benefit should also be measured. Thefirst subjects for human treatment studies mayhave more severe disease, in which vision is al-ready substantially compromised. Each patientshould be carefully monitored to ensure thatthere is no detrimental effect. Then individualswith more moderate disease might be consid-ered for treatment. In designing a human study,clinical examination methods that identify un-derlying cellular dysfunction should be used, ifpossible. In a Phase I safety study, these mightinclude reading evaluation to monitor visualacuity, degree of ERG impairment, and OCTimaging to monitor the structure of the retina.The principles learned from genetic analysesin XLRS patients and efficacy studies in XLRSmice support clinical testing of a RS1 gene ther-apy approach to reduce vision loss in retinoschi-sis patients.

ACKNOWLEDGMENTS

The authors are grateful to Dr. Catherine Cuk-ras for the fundus photo and OCT scan from an

XLRS patient and Cindy Best, Matthew McMa-hon, and Santa Tumminia for their assistance inthe preparation of this review. Supported by theNational Institutes of Health (NIH) IntramuralResearch Program through the National Insti-tute on Deafness and Other CommunicationDisorders and the National Eye Institute.

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