Gene therapy prevents photoreceptor death andpreserves retinal function in a Bardet-Biedlsyndrome mouse modelDavid L. Simonsa,1, Sanford L. Boyeb, William W. Hauswirthb, and Samuel M. Wua

aDepartments of Ophthalmology and Neuroscience, Baylor College of Medicine, Houston, TX 77030; and bDepartment of Ophthalmology, University ofFlorida, Gainesville, FL, 32610

Edited by John E. Dowling, Harvard University, Cambridge, MA, and approved March 7, 2011 (received for review December 23, 2010)

Patients with Bardet-Biedl syndrome (BBS) experience severeretinal degeneration as a result of impaired photoreceptor trans-port processes that are not yet fully understood. To date, there isno effective treatment for BBS-associated retinal degeneration,and blindness is imminent by the second decade of life. Here wereport the development of an adeno-associated viral (AAV) vectorthat rescues rhodopsin mislocalization, maintains nearly normal-appearing rod outer segments, and prevents photoreceptor deathin the Bbs4-null mouse model. Analysis of the electroretinogram a-wave indicates that rescued rod cells are functionally indistinguish-able fromwild-type rods. These results demonstrate that gene ther-apy can prevent retinal degeneration in a mammalian BBS model.

ciliopathies | intra-flagellar transport | electroretinography

Bardet-Biedl syndrome (BBS) is clinically diagnosed by thepresence of at least four of the following signs: retinal dys-

trophy, polydactyly, obesity, learning disabilities, male hypo-gonadism, and renal anomalies (1). Of these, the visual phenotypeis particularly devastating: the average BBS patient will progressto legal blindness before his or her 16th birthday. There are cur-rently 15 genetic loci (Online Mendelian Inheritance in Man#209900) that are known to be associated with BBS, and althoughinheritance of this disease was once thought to follow a classicautosomal recessive pattern, recent evidence suggests that a morecomplex pattern, termed “triallelic inheritance,” is involved atcertain BBS loci (2). Only within the last decade has evidenceemerged that BBS proteins play a role in ciliary function (3).Morespecifically, Bbs4 and the other BBSome components seem tobe involved in both recruitment of cargo toward the ciliary basalbody (4, 5) and in intraflagellar transport along the cilium (5–7).Several reports have elucidated the mechanisms by which dis-ruption of BBS proteins give rise to the individual phenotypesseen in this highly pleiotropic syndrome (8–11).Retinal degeneration is a central feature of all BBS mouse

models generated to date (8, 12–16). In rod and cone photo-receptors, the connecting cilium is a highly specialized ciliarystructure that serves as the sole conduit from the inner segment tothe outer segment. Because protein synthesis occurs proximal tothe outer segment, rhodopsin and other visual proteins must betrafficked through the connecting cilium to reach their site of actionin the outer segment. Abd-El-Barr et al. (11) showed that whenBbs4 was deleted in mice, rhodopsin and cone opsins becamegrossly mislocalized in rod and cone photoreceptors, respectively.This was followed by apoptotic photoreceptor death and de-terioration of the electroretinogram (ERG) a- and b-waves (14).Ultrastructural analysis of rods from young animals revealednormal-appearing connecting cilia and basal body structures. Thislatter finding has important implications for gene therapy: becausethe structural transport apparatus seems to develop normally inBbs4-null photoreceptors, we hypothesized that postnatal supple-mentation of the Bbs4 gene in rods would rescue rhodopsin mis-localization and prevent rod photoreceptor death.

To test our hypothesis, we chose a pseudotyped adeno-associatedvirus (AAV) as a delivery vector. AAV has many desirable fea-tures as a retinal gene therapy vector, including low immunoge-nicity (17), high transduction efficiency (18), long-term transgeneexpression (19), and specific serotype-dependent cellular trop-isms (20). Furthermore, data from three landmark phase I clinicaltrials suggest that AAV has an excellent safety profile in humans(21–23). Here, we describe the results of AAV-mediated Bbs4delivery into the rods ofBbs4-null mice. Our findings demonstratethat gene therapy can rescue rhodopsin mislocalization in thisBBSmouse model and maintain nearly normal rod outer segmentmorphology. Moreover, our treatment prevents photoreceptordeath, improves electrophysiological function of the retina,and preserves visually evoked behavioral responses. These datafrom a mammalian model suggest that BBS-associated retinaldegeneration can be treated.

ResultsRetinal Degeneration in the Bbs4-Null Mouse. We performed ana-tomical and functional studies in Bbs4-null mice and wild-typelittermates to obtain baseline values before our rescue experi-ments. In the wild-type retina, immunohistochemical rhodopsinstaining (red) localized to the outer segment region of rod pho-toreceptors and was completely absent from the outer nuclearlayer (ONL) (Fig. 1A, 8 wk of age). Rhodopsin was also found inthe outer segment region of age-matched Bbs4-null (−/−) retinas,but this was accompanied by mislocalized rhodopsin moleculesthat tended to form annular patterns (Fig. 1B, arrowheads)around individual photoreceptor nuclei (blue). Fig. 1C illustratesthe progressive loss of photoreceptor nuclei in the BBS retina,which others have shown to occur through apoptosis (11, 12). At 2wk of age the −/− retina already showed evidence of photoreceptorloss (seven to eight nuclei per ONL column) compared with anage-matched +/+ control whose ONL was 10 to 11 nuclei thick.Photoreceptor death in the −/− retina progressed steadily overtime, and only one to two nuclei per ONL column remained at 16wk. Outer segments in the −/− retina were shorter and more het-erogeneous than +/+ outer segments at the earliest time pointsexamined and were entirely unrecognizable by 16 wk.We performed ERGs to measure retinal function in Bbs4-null

mice over time. At all ages and flash intensities examined, a- andb-wave amplitudes were severely attenuated in −/− mice (redtraces) compared with +/+ controls (black traces). Representa-

Author contributions: D.L.S. and S.M.W. designed research; D.L.S. performed research;D.L.S., S.L.B., and W.W.H. contributed new reagents/analytic tools; D.L.S. and S.M.W.analyzed data; and D.L.S. wrote the paper.

Conflict of interest statement: W.W.H. and the University of Florida have a financial in-terest in the use of adeno-associated virus therapies and own equity in a company (AGTCInc.) that might, in the future, commercialize some aspects of this work.

This article is a PNAS Direct Submission.1To whom correspondence should be addressed. E-mail: dlsimons@bcm.edu.

This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10.1073/pnas.1019222108/-/DCSupplemental.

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tive 8-wk-old data are shown for rod-only (Fig. 1D) and mixedrod–cone (Fig. 1E) responses. To parameterize the rod photo-responses, we performed an ensemble fit of the Cideciyan-Jacobson photocurrent model (24) against ERG a-wave data fromtwo different flash intensities. Fig. 1F shows the a-wave leadingedges from a representative −/− eye (Right) whose rod response islower in both amplitude and sensitivity compared with an age-matched wild-type control eye (Left).

Expression of AAV Transgenes. Previous characterization of thismouse model has shown that cone opsin mislocalization is severeby 2 wk of age (11), and cone ERGs are attenuated by 88% at 4 wk(14). For these reasons, we chose to direct our gene therapyefforts toward rescue of rod photoreceptors, which have a slowercourse of degeneration.Weused a pseudotyped self-complementaryadeno-associated virus (scAAV5) containing the mouse opsin

promoter (mOP) to drive strong expression in rods (25). Two viralconstructs were used in this study: a control virus containing GFP(henceforth referred to as scAAV5-mOP-GFP) and a therapeuticvirus containing the mouse Bbs4 gene with an N-terminal HAtag (scAAV5-mOP-BBS4). In our hands, subretinal injection of1.1 μL of scAAV5-mOP-GFP into a wild-type mouse eye resultedin a localized region of GFP expression (Fig. 2A) that covered4.8% ± 1.1% of the retina (mean ± SEM, n= 6). Transduction ofrod cells within those regions was extremely efficient, with nearly100% of rod cells expressing GFP close to the injection site (Fig.2Bi). In contrast, cone photoreceptors (arrowheads in Fig. 2Bi,red signal in Fig. 2Bii) expressing GFP were present but rare.Cones were identified by examining serial optical sections fromflat-mounted retinas stained with rhodamine-conjugated peanutagglutinin (PNA) (Movie S1).Injection of our scAAV5-mOP-BBS4 construct into the sub-

retinal space resulted in a single HA-immunoreactive band byWestern blot (Fig. 2C). This band, which was absent in the unin-jected control sample, was located at ≈59 kDa, the expected mo-lecular mass for Bbs4 (UniProt# Q8C1Z7) with an HA tag. Todetermine subcellular localization of the viral Bbs4 protein, weperformed immunohistochemistry on 40-μm vibratome sections.Retinas injected with scAAV5-mOP-BBS4 demonstrated a clearband of punctate HA-positive staining (asterisk) that was not seenin uninjected control retinas (Fig. 2D). This band of staining wasmost dense in the inner segment region located between the pho-toreceptor nuclei (ONL) and the rhodopsin-rich outer segments(Fig. 2D, Right); however a few punctae were observed at theproximal margin of the outer segment layer. These data are con-sistent with tissue localization of the endogenous protein (4, 11) aswell as observations from cell culture experiments that BBS4 can befoundboth inside the cilium(5) and at the centriolar satellites (4, 5).

Rescue of Rhodopsin Mislocalization. After establishing the sub-cellular localization of viral Bbs4, we next determined whether ourgene therapy treatment could prevent rhodopsin mislocalization.Two-week-old Bbs4-null mice were treated by subretinal injectionof scAAV5-mOP-BBS4 into the temporal hemiretina of one eye.At 12 wk of age, horizontal cryostat sections were cut that con-tained a virus-treated region in the temporal half of the sectionand an untreated control region in the nasal half. Immunohisto-chemistry indicated that the percentage of photoreceptors withannular mislocalized rhodopsin staining (arrowheads) was lowerin treated regions (Fig. 3A) compared with untreated regions (Fig.3B). The percentages in treated vs. untreated regions for the threeretinas examined (Fig. 3C) were 0.30% vs. 4.19% (P < 0.0001),0.23% vs. 1.58% (P < 0.01), and 0.09% vs. 2.30% (P < 0.0001). Atleast 600 ONL cells were counted in each region, and significancewas calculated using a χ2 test for a 2 × 2 contingency table.Outer segments in Bbs4-null retinas also showed improvement

after treatment with scAAV5-mOP-BBS4. Treated −/− micekilled at 16 wk demonstrated patches of dense, homogeneousouter segments in the temporal hemiretina measuring 20.7 ±0.52 μm in length (mean ± SEM, n = 12) (Fig. 3D, Right). Thesewere similar to wild-type outer segments (Fig. 3D, Left) in bothappearance and length (22.6 ± 0.21 μm, n= 12). Outer segmentsfrom untreated −/− retinas, in contrast, were sparse, heteroge-neous, and short (13.1 ± 0.44 μm, n = 12) at 4 wk and un-recognizable at 16 wk (Fig. 3D, Center Left and Center Right).

Photoreceptor Rescue. To determine whether gene therapy couldprevent photoreceptor cell death in the Bbs4-null retina, wecounted the number of nuclei remaining in cellular columns ofthe ONL. Fig. 4 shows both eyes from a −/− mouse treated at 2 wkand killed at 16 wk. The left eye received a temporal scAAV5-mOP-BBS4 injection, whereas the right eye received a temporalscAAV5-mOP-GFP injection, leaving the nasal portions of bothretinas unexposed to either virus. Heat maps of ONL thickness

Fig. 1. Rhodopsin mislocalization and retinal degeneration in Bbs4-nullmice. Immunolocalization of rhodopsin (red) in 8-wk-old wild-type (A) andBbs4−/− (B) retinas. Topro-3 (blue) marks nuclei, and PNA (green) identifiescone sheaths. Arrowheads in B indicate rod photoreceptors that exhibitmislocalized rhodopsin. (C) Photomicrographs illustrate the progressive lossof photoreceptors in the ONL of Bbs4−/− retinas, with wild-type sections atboth ends for comparison. (D–F) Attenuation of the ERG accompaniesphotoreceptor loss. At 8 wk, Bbs4−/− (red) responses are much smaller thanwild-type littermates (black) at flash intensities eliciting either rod-only (D)or mixed rod–cone (E) responses. F is a high-magnification view of a-waveleading edges at two different intensities, with a photoresponse modelfitted to the data (dotted traces). Flash intensities in log scot cd*s/m2 are−3.65, −2.93, −2.08, and −0.89 forD; 3.45 for E; 2.10 and 3.45 for F. (Scale bars,20 μm in A and B, 10 μm in C.)

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(Fig. 4A) clearly demonstrate a localized region in the temporalhalf of the left eye, where the ONL was four to six photoreceptornuclei in thickness. This region corresponds precisely to theportion of the retina exposed to the therapeutic Bbs4 virus.Regions not receiving the therapeutic virus (i.e., the nasal half of theleft eye and both halves of the right eye) had undergone un-mitigated degeneration that left the photoreceptor layer only one totwo nuclei thick. Photomicrographs (Fig. 4B) from the five locationsindicated in the heat maps illustrate the stark contrast between −/−

retina with and without gene therapy treatment. Treated regions(i and ii) have a much thicker ONL along with nearly normal-appearing outer segments, whereas untreated regions (iii–v) havea homogeneously thin ONL and no outer segments.The pattern of localized ONL thickness in the region treated

with scAAV5-mOP-BBS4 was consistent across all retinas ex-amined. This included two animals killed at 12 wk (Figs. S1 andS2), two animals killed at 16 wk (Fig. 4 and Fig. S3), and one

animal killed at 45 wk (Fig. S4). This oldest animal was of par-ticular interest because the untreated eye contained absolutelyno photoreceptors, whereas the temporal region of the treatedeye had six to seven rows of photoreceptors at its thickest point.

Functional Improvement of Vision. To assess rod function in theBbs4-null retina after gene therapy, we performed ERGs andparameterized a-wave responses using least-squares fit of theCideciyan-Jacobson photocurrent model (Eq. 1). This analysisproduced an amplitude (Rmax) and sensitivity (σ) value to de-scribe the rod response of each eye. Fig. 5A shows actual a-wavedata (solid traces) at two different intensities and fitted curves(dashed traces) for a 12-wk-old −/− mouse with one eye treated(Left) and the other untreated (Right).After treatment at 2 wk of age, ERG a-waves were analyzed at

4, 8, 12, and 16 wk. Data are displayed in Fig. 5B, with both in-dividual eyes (open symbols) and average values (filled symbols)shown. At all four time points, treated eyes (scAAV5-mOP-BBS4) on average had both higher amplitude and higher sensi-tivity than control eyes (scAAV5-mOP-GFP or uninjected). Thedifference between treated and control eyes was small at 4 wk butgrew wider and reached statistical significance at all three sub-sequent time points (Table 1). Of particular interest is the factthat the average sensitivity of treated eyes actually increased from12 to 16 wk, reaching a value of 3.80 ± 0.077 log scot cd−1m2s−3

(mean ± SEM, n= 15). This value was virtually indistinguishablefrom the age-matched wild-type value of 3.83 ± 0.064 (n = 6).Average values of Rmax and σ from Fig. 5B were substituted intoEq. 1 to produce average a-wave leading edges for both groups

Fig. 2. Transgene expression after subretinal injection of AAV. (A) Retinalflatmount 4 mo after scAAV5-mOP-GFP injection revealed localized geneexpression only near the site of injection. (Scale bar, 1 mm.) (Bi) Confocalimage of the top of the ONL showing rods (green from GFP) and cones (darkcells marked by arrowheads). Cones were identified by rhodamine-PNAstaining in serial optical sections. (Scale bar, 10 μm.) (Bii) Inner segmentsbelonging to three cones (PNA) are the only ones not expressing GFP. (Scalebar, 5 μm.) (C) Western blot analysis of a retinal sample injected with AAVcarrying the HA-Bbs4 construct shows an HA-immunoreactive band near 59kDa that is absent in the uninjected control sample. (D) AAV-mediated Bbs4expression localizes primarily to the inner segments and proximal outersegments (asterisk), whereas no such staining is seen in uninjected controls.(Scale bar, 20 μm.) Right: A rhodopsin signal is shown to delineate the OS/ISborder. All experiments were performed in phenotypically normal controls(Bbs4+/+or Bbs4+/−). Topro-3, nuclear stain; PNA, cone sheath marker; OS,outer segment; IS, inner segment; OPL, outer plexiform layer.

Fig. 3. scAAV5-mOP-BBS4 rescues rhodopsin mislocalization and restoresouter segment morphology. Treated (A) and untreated (B) regions of thesame retina demonstrate clear differences in the percentage of photo-receptors with mislocalized rhodopsin (arrowheads). The red signal imme-diately below the ONL represents nonspecific staining characteristic of thisantibody (Fig. 1A). Note also that the ONL is considerably thicker in thetreated retina (A). Red, rhodopsin; blue, nuclear stain Topro-3. (Scale bars, 20μm.) (C) In all three retinas examined at this age, the percentage of pho-toreceptors exhibiting rhodopsin mislocalization was significantly lower intreated vs. untreated regions (*P < 0.01; **P < 0.0001; significance calcu-lated by χ2 test for a 2 × 2 contingency table). (D) Regions of Bbs4−/− retinatreated with scAAV5-mOP-BBS4 contained outer segments that were strik-ingly similar to WT outer segments. In untreated control Bbs4−/− retina,outer segments are not recognizable at 16 wk of age. (Scale bar, 10 μm.) OS,outer segment; IS, inner segment; INL, inner nuclear layer.

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(Fig. 5C) at all four time points (4 wk Left, 16 wk Right). Note thefaster rise time and greater amplitude of the treated (blue) tracesat all times. Interestingly, the amplitude difference betweentreated and control remained relatively constant over time, eventhough both traces decreased in absolute amplitude (Discussion).We also performed optokinetic response (OKR) testing to

determine whether the limited region of preserved retina wassufficient to elicit a visually evoked behavioral response. A totalof nine mice were tested (ages 9–11 mo), each animal havingreceived a monocular scAAV5-mOP-BBS4 injection at 2 wk ofage. Preliminary testing under scotopic conditions revealed thatfour of the nine treated eyes could elicit a positive OKR, whereasno such response was seen for any of the contralateral untreatedeyes. Further behavioral testing is currently under way.

DiscussionHere we present evidence that gene therapy can prevent themajorpathologic hallmarks that are found in theBbs4-null retina (11, 13,14). Specifically, viral Bbs4 expression in rod photoreceptors res-cues rhodopsin mislocalization and maintains healthy-appearingrod outer segments. Photoreceptor death is prevented in regionsreceiving gene therapy treatment, whereas untreated regionsdegenerate rapidly. We confirmed these anatomical findings byelectroretinography and behavior. ERG rod responses demon-strated greater amplitude and higher sensitivity in treated eyes vs.untreated controls, and positive OKRs were elicited from sometreated eyes but no untreated controls. Collectively, these resultssuggest that gene therapy is a feasible approach for treatingblindness in human BBS patients. On the basis of our success insupplementing the Bbs4 gene, it seems very likely that other formsof BBS, especially those that are molecularly linked to theBBSome (5), will also be suitable targets for gene therapy. Thesame may hold true for other retinal ciliopathies, including Ushersyndrome, Joubert syndrome, Senior-Loken syndrome, and cer-tain forms of Leber congenital amaurosis (26). Furthermore, be-cause obesity in BBS is also thought to occur by an analogousproteinmislocalizationmechanism involving the leptin receptor inhypothalamic neurons (9), it is possible that some forms of syn-dromic obesity may also be candidates for gene therapy.In our hands, subretinal injection of scAAV5-mOP-GFP

resulted in GFP expression with an average retinal coverage ofonly 4.8%. Although this is less area than others have estimated,we achieved extremely high rod transduction within the coveragearea (approaching 100% near the site of injection) andminimized

adverse events resulting from mechanical trauma to the tissue.The viral coverage pattern was beneficial for our study: it providedconvenient internal controls when examining treated (temporal)and untreated (nasal) regions of transverse retinal sections.Moreover, because the transduction of rod cells was nearly 100%locally and absent in distant regions, the histological effects of thevirus were readily apparent. For reasons previously mentioned,we chose the mouse opsin promoter to focus our efforts on res-cuing rod photoreceptors. As a result, our therapeutic viral con-struct led to clear improvement in the rod system but little coneimprovement. However, given a wider therapeutic time windowand an appropriate promoter (27–29), there is no obvious reasonwhy cones could not also be rescued. Unlike the mouse used inthis study, the Bbs1 M390R knockin has relatively normal coneERG function at 11 wk of age (16) and may therefore be an ap-propriate model for future evaluation of cone rescue.Although the precise molecular role of Bbs4 and other BBS

proteins in photoreceptors is not completely understood [seeZaghloul and Katsanis (30) for a review], much of the literaturepoints to a role in intracellular protein trafficking and a localiza-tion in the vicinity of the ciliary basal body. Our observation ofviral Bbs4 expression in the inner segment and proximal outersegment is entirely consistent with this idea. ScAAV5-mOP-BBS4was able to reverse the prototypical rhodopsin mislocalizationphenotype seen in several BBS mouse models (11, 12, 15), as wellas othermodels in which ciliary proteins were deleted (31, 32). It isnot entirely clear whether rhodopsin mislocalization in this modelis a direct result of Bbs4 deletion or rather a secondary insultcaused by photoreceptor degeneration. This distinction, however,is of little consequence for the present study. Rhodopsin mis-localization was found to precede apoptotic photoreceptor deathin the Bbs2- and Bbs4-null mouse models (11, 12), so on the basisof our finding that gene therapy rescues rhodopsinmislocalization,we fully expected that gene therapy would also prevent photore-ceptor death. This was indeed the case: localized injection ofscAAV5-mOP-BBS4 at 2 wk of age resulted in localized photo-receptor rescue that persisted even until the last time pointexamined (45 wk). Conversely, untreated regions steadily degen-erated until photoreceptor loss was complete. We have thereforeestablished that a therapeutic window exists in the Bbs4-nullmouse, during which the retinal disease process is reversible.Analysis of the full-field ERG a-wave using the Cideciyan-

Jacobson model (24) proved to be remarkably sensitive in thisstudy.Without having to use a multifocal ERG technique we were

Fig. 4. Treatment with scAAV5-mOP-BBS4 prevents photo-receptor death. (A) At 16 wk, photoreceptor survival wasassessed by outer nuclear layer (ONL) thickness in horizontalsections containing optic nerve. Heat maps indicate thickouter nuclear layer (red) at the site of scAAV5-mOP-BBS4injection, and thin outer nuclear layer (blue) in all otherregions. (B) Photomicrographs from treated (i and ii) anduntreated (iii–v) locations indicated in A. (Scale bar, 10 μm.)Outer segments in Bii became bent during fixation and ap-pear in cross-section. IS, inner segment; OS, outer segment;INL, inner nuclear layer.

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able to observe a highly significant (P < 0.0001) improvementin rod function even though our subretinal injections coveredonly 5% of the retina. The corneal ERG potential is a summationof all radial extracellular currents in the eye, so in our cohort ofeyes treated with scAAV5-mOP-BBS4 we hypothesized thatthe ERG represents a linear combination of 5% healthy (treated)retina and 95% degenerating (untreated) retina. The maximala-wave amplitude, Rmax, is related to the total circulating darkcurrent in the outer segments at the time of light onset (33) and istherefore expected to decline in this mouse model as outer seg-ments are lost owing to photoreceptor death. Our a-wave data(Fig. 5C) show that Rmax in treated eyes declines from 4 to 16 wkbut is larger than Rmax from untreated control eyes at every timepoint. More importantly, the amplitude difference between the

two experimental groups is nearly constant. The constant differ-ence suggests that treated eyes have a small population of rescuedouter segments that remain healthy over time (Fig. 4 Bi and Bii)and steadily contribute to the a-wave. This, however, is super-imposed on a large background signal originating from unhealthyouter segments that degenerate over time.Our linear summation hypothesis is also supported by changes

in the a-wave sensitivity (σ) over time. Shady et al. (34) con-cluded from human retinitis pigmentosa patients that rod de-generation resulted in progressive reduction of transductionamplification, a quantity closely related to the sensitivity param-eter reported in the present study. We show that rod sensitivity inuntreated Bbs4-null eyes decreases monotonically from 4 to 16wk. In treated eyes, however, sensitivity decreases from 4 to 12 wk,followed by a notable increase from 12 to 16 wk. It is of particularinterest that the sensitivity of treated eyes at 16 wk is very similarto the sensitivity of age-matched wild-type eyes. In the context ofour linear summation model, these data suggest that the jump insensitivity between 12 and 16 wk represents the gradual disap-pearance of the 95% unhealthy rod signal and an emergingdominance of the 5% healthy (treated) rods with relatively nor-mal rod phototransduction parameters. The preservation of vi-sually evoked behavioral responses from four of nine treated eyesfurther confirms that a population of functional rods survived asa result of our treatment. The fact that any treated mice exhibitedthese behavioral responses was somewhat surprising, consideringthe limited area of rescued photoreceptors. Further studies arecurrently under way to correlate anatomical, electrophysiological,and behavioral rescue at the level of individual eyes.From an experimental perspective our mean coverage factor

of 5% per viral injection was more than sufficient to observeanatomical and functional benefit, thus validating the efficacy ofour viral construct. In large animal models, retinal coverage of20–30% is readily achievable by subretinal injection (35, 36). Iftranslated to a human subject, 30% coverage containing themacular region would have the potential to preserve nearlynormal visual acuity. An important prerequisite for gene therapy,however, is a timely genetic diagnosis before extensive photo-receptor death has occurred. Clinical studies suggest that amongBBS1 patients, only half will have visual acuity of 20/60 or betterat the time of diagnosis; the situation seems to be worse for thosewith BBS10 (37, 38). This underscores the importance of BBSawareness in the ophthalmology and broader medical commu-nities. Our results are an encouraging first step toward preservingvision in BBS patients, but ultimate success of such a therapy willrequire a collaborative effort between translational researchersand vigilant clinicians.

Fig. 5. Analysis of ERG a-wave after scAAV5-mOP-BBS4 treatment. (A)Representative 12-wk a-wave leading edges and fitted model traces (dottedlines) for treated (Left) and control (Right) eyes of a Bbs4−/− mouse. (B) Themodel-fitting procedure returned amplitude (Rmax) and sensitivity (σ)parameters for eyes tested at 4, 8, 12, and 16 wk. Individual eyes are shownas open symbols (blue, scAAV5-mOP-BBS4; red, scAAV5-mOP-GFP or no in-jection), and filled symbols represent averages (error bars = SEM). Groupaverages are statistically different at 8, 12, and 16 wk (Table 1). (C) AverageRmax and σ values from B were used as inputs to the photocurrent model,and output traces are shown for each of the four time points examined.

Table 1. Electroretinogram a-wave analysis

Age (wk) Cohort Rmax (μV)σ (log scotcd−1m2s−3) n P

4 Treated 119.4 ± 11.4 3.66 ± 0.075 12 0.583Control 105.8 ± 7.63 3.61 ± 0.046 16

8 Treated 72.9 ± 4.97 3.62 ± 0.040 18 0.003Control 59.4 ± 3.07 3.42 ± 0.041 22

12 Treated 42.6 ± 3.45 3.60 ± 0.061 20 <0.0001Control 31.1 ± 1.62 3.25 ± 0.044 24

16 Treated 28.5 ± 2.91 3.80 ± 0.077 15 <0.0001Control 17.5 ± 1.60 3.08 ± 0.100 21

Rod response amplitudes (Rmax) and sensitivities (σ) from Bbs4−/− eyesinjected at 2 wk of age. Treated eyes received scAAV5-mOP-BBS4, and con-trol eyes received either scAAV5-mOP-GFP or no injection. P values werecalculated using Hotelling’s t-squared statistic.

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Materials and MethodsViral Constructs. Full-length cDNA for the mouse Bbs4 gene (accession no.BC145771) was obtained from Open Biosystems. An HA epitope tag(YPYDVPDYA) was added to the N terminus of Bbs4, followed by ligation ofHA-Bbs4 into the sc-MOPS500-hGFP plasmid in place of hGFP. The plasmidtransfection method using HEK293 cells as previously described (39) wasused to produce and purify scAAV2/5 vectors carrying either Bbs4 or GFP.Viral titers were determined by real-time PCR as previously described (40)and were 6.82 × 1012 VG/mL for scAAV5-mOP-BBS4 and 3.64 × 1012 VG/mLfor scAAV5-mOP-GFP.

Animals and Subretinal Injections. Bbs4-null mice were generated as pre-viously described (8). All animal procedures were approved by the In-stitutional Animal Care and Use Committee at Baylor College of Medicine.Details of the transscleral subretinal injection technique are given in SIMaterials and Methods.

Histology and Immunoblotting. Methods were performed as previously de-scribed (11) with minor modifications. Ten-micrometer frozen sections werestained with 1:1,000 anti-rhodopsin mAb (RET-P1 clone; Neomarkers) andDAPI. For HA-BBS4 identification, a 1:1,000 dilution of anti-HA mAb wasused (16B12 clone; Covance). For light microscopy, 1-μm Epon sections werestained with methylene blue and basic fuchsin. To create heat map repre-sentations, digitized images were evenly divided into 32 regions per hemi-retina. The thickness value for each region was determined by averagingthree counts of nuclei found in an ONL column.

Functional Vision Testing.Mice were anesthetized by i.p. injection of 51 mg/kgketamine, 10 mg/kg xylazine, and 0.86 mg/kg acepromazine, and ERGs wererecorded as previously described (11). A-wave leading edges were modeledusing the method described by Cideciyan and Jacobson (24). Briefly, dark-adapted ERGs were recorded in response to two bright flashes (2.10 and 3.45log scot*cd*s/m2), and the following equation was fit to both curves si-multaneously using least-squares minimization:

RðI; tÞ ¼ Rmax

�1− exp

�−12Iσ��

ε2 − 6ετþ 12τ2�− e

−ετ�ε2 þ 6ετ

þ 12τ2���

∗e−

ετrm [1]

where I is the flash intensity; Rmax, the maximum response amplitude; σ,sensitivity; and t, time after stimulus onset. More information about Eq. 1,curve fitting software, and OKR testing can be found in SI Materialsand Methods.

ACKNOWLEDGMENTS.We thank Zhuo Yang and Ralph Nichols for help withanimal husbandry and histology, respectively; Jim Lupski, M.D., Ph.D., forkindly providing the mouse model; and Ben Frankfort, M.D., Ph.D., forcarefully reading the manuscript. Support for this work was provided byNational Institutes of Health Grants EY019908, EY04446, and EY02520 (toS.M.W.) and EY13729 and EY08571 (to W.W.H.); Retina Research Foundation(Houston) (S.M.W.); Macular Vision Research Foundation, Foundation Fight-ing Blindness, Eldon Family Foundation, and Vision for Children (W.W.H.);Research to Prevent Blindness, Inc. (S.M.W. and W.W.H.); and the NationalScience Foundation GK-12 Fellowship and Baylor Medical Scientist TrainingProgram (D.L.S.).

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