`
`

`

`WOHpapRajuMeg
`
`
`oo
`al
`mi
`
`ua=4o
`
`O
`oO
`
`mw
`
`oA 3O
`
`o
`
`x&2&==
`
`> 2g
`
`S 2o
`
`a
`
`
`
`AAV2 Gene Therapy Readministration in Three
`Adults with Congenital Blindness
`Jean Bennett,
`Manzar Ashtari,?*"
`Jennifer Wellman,’ Kathleen A. Marshall,”
`Laura L. Cyckowski,? Daniel C. Chung,’ Sarah McCague,” Eric A. Pierce,'** Yifeng Chen,”
`Jeannette L. Bennicelli,' Xiaosong Zhu,* Gui-shuang Ying,’ Junwei Sun,’ J. Fraser Wright,*
`Alberto Auricchio,®” Francesca Simonelli,°* Kenneth $. Shindler,’ Federico Mingozzi,*
`Katherine A. High,”? Albert M. Maquire’**
`
`L2gt
`
`3yt
`
`Demonstration of safe and stable reversal of blindness after a single unilateral subretinal injection of a recombinant
`adeno-associated virus (AAV) carrying the RPEGS gene (AAV2-hRPE65v2) prompted us to determine whetherit
`was possible to obtain additional benefit through a second administration of the AAV vector to the contralateral
`eye. Readministration of vector to the second eye was carried out in three adults with Leber congenital amaurosis
`due to mutations in the RPE6S gene 1.7 to 3.3 years after they had received their initial subretinal injection of
`AAV2-hRPEGSv2. Results (through 6 months} including evaluations of immune response, retinal and visual function
`testing, and functional magnetic resonance imaging indicate that readministration is both safe and efficacious
`after previous exposure to AAV2-hRPEGSv2.
`
`TASPWSSW ENE SohPSSA“ gs
`SWE NASRS USL FASE
`
`Leber congenital amaurosis (LCA) is a group ofhereditary retinal
`dystrophies characterized by profound impairment in retinal and vi-
`sual function in infancy and early childhood followed by progressive
`deterioration and loss of retinal cells in the first few decades oflife
`(J~3). LOA is usually inherited as an autosomal recessive trait, and
`mutations in 15 different genes have been reported so far (4, 5). One
`of the more commonforms of LCA, LCA2, is due to routations in the
`
`RPEOS gene (6, 7). This gene encodes an all-trans-retinyl ester isom-
`erase, an enzyme critical to the function of the retinoid cycle (8, 9).
`Without RPE6S, very little 11-cis-retinal, the vitamin A derivative that
`is the chromophore of rod and cone photoreceptor opsins, is made
`(8, 9}. Without P1-cis-retinal, opsins cannot capture light and relay this
`into electrical responses to initiate vision (8, 10). Successful proof-of
`principle studies in LCA? murine and canine animal models using a
`replication-defective adeno-associated viral vector @AAV) (1-14
`demonstrated that the biochemical blockade of the visual cycle due
`to RPE6S deficiency could be overcome through gene augmentation.
`Safety and dosing studies in large animals then provided the pre-
`
`BST!
`3t
`
`italy. "Medica
`“Department of Ophthmology, Seeconda
`
`5, 80131
`di Napoli, Via S. Pansini 5, 80134 Naples, italy.
`°
`vic Center Boulevard,PI
`1
`
`}
`
`
`5"Oecular Genomics Institute, Massachusetts
`@
`Boston, MA 02114, USA
`
`clinical safety and efficacy data that formed the impetus to test this
`approach in human clinical trials (15-17).
`We reported safe and stable amelioration in retinal and visual
`functionin all 12 patients treated in a phase1/2 study at The Children’s
`Hospital of Philadelphia (CHOP) (16, 18-20). These individuals had
`been injected subretinallyin the eye with worse vision ina dose-escalation
`studywith doses ranging from1.5 x 10" to 1.5 x 10"! vector genomes
`(vg) ofthe AAV2 vector carrying the RPE6S gene (AAV2.hRPE65v2}
`(16, 18). Each oneof the subjects showed improvement in multiple mea-
`sures ofretinal and visual function in the injected eve. Most of the
`subjects showed improvementin full-field light sensitivity and pupil-
`lary light reflex (PLR). About halfof the subjects showed significant
`improvement in visual acuity, and all showed a trend toward improve-
`
`ment in visual fields. Five of the 12 patients Gnchidingall pediatric sub-
`jects age 8 to 11 years) developed the ability to navigate a standardized
`obstacle course (16, 18). The improvements were observed as early as
`i month after treatment and persisted through the latest time point
`
`(now4 years for theinitial subjects) (76, 18, 20}. Functional magnetic
`resonance imaging ((MRD studies carried out in subjects after they had
`
`received the injection also showed that the visual cortex became re-
`sponsive to retinal input after this unilateral gene therapy, even after
`prolonged visual deprivation (20). Both the retina and the visual cortex
`became far more sensitive to dim light and lower-contrast stimuli.
`The success ofthe unilateral injections begged the question ofwhether
`additional visual function could be further gained in the contralateral
`eye of these patients. Because the immune consequences of subretinal
`readrninistration of rAAV2 were unknown, we carried out contralateral
`eye readministration studies in two different large-animal models. Re-
`administration resulted in efficacy in both eyes in the affected dogs and
`appeared safe in both affected dogs and unaffected nonhurnan primates
`ay tre.
`og ae ety nt.
`(2). However, there is little precedent for the ability to safely re-
`administer rAAV in harnans and obtain a therapeutic effect. There
`
`diminish the benefits that the subjects had obtained in their previously
`injected eye. Wetherefore proceeded cautiouslyto test safety and effi-
`cacy of administration to the contralateral eye in three adult subjects
`
` was also a concern that immuneresponses after readrministration would
`
`
`
`ins.org
`
`8 February 2012
`
`Vol 4 Issue 120 120rai5
`
`4
`
`

`

`+
`f
`re
`
`Th
`
`
`
`who had already underggoneuniilateral subretinal injection in our phase
`1/2 dose-escalationstudy (16, 18).
`and postsurgical testing, we demon-
`Through comparison of pre-
`atrate that delivery of AAV2-hRPE65v2 to the contralateral eye is safe
`evenif years have passed since the initial treatment. Further, before and
`afler comparisons of psychophysical data and {MRI results provide ad-
`ditional evidence for theeffectiveness of gene therapy reaciministration
`in LCA2 patients and also reveal the magnitude and pattern of im-
`provement. Results in two patients receiving different doses in each eye
`
`suggest a possible dose-response effect of the gene therapyvector.
`
`Follow-on enroliment and study design
`The readministration study was carried out as a “follow-on” (FO)
`study to the original phase 1/2 protocol (NCTO1208389). The original
`protocol entailed injection into each subject's more impaired eye (16, 13}.
`‘TheInstitutional Review Board (IRB) had given approval for thecontra-
`lateral eye administration as longas the first three subjects were adults.
`
`Thefirst three adults enrofled in the FO study were CH12, CHIL, and
`NPOL, all of whomhave missense mutations in RPE655 (Table 1), and
`these individuals self-selected on the basis of availability. The disease
`was advanced in each one of these subjects, the degree of which
`correlated with their age due to the degenerative nature of LCA2. These
`individuals had received their initial injection 1.7 to 3.4 years earlier and
`were enrolled sequentially (with an 8-week interval between each enroll-
`ment). After providing informed consent, the subjects underwent “FO
`baseline” immunological and retinal/visual testing before the readmin-
`istration. The schedule of tests in the FO study wassimilar to but not
`identical to the schedule in the initial study (table $1). Some tests that
`had been usedin the initial study were dropped (for example, electro-
`retinograms). Other analyses had been added during the course ofthe
`initial study, and these were maintained in the FO studyincluding the
`full-Beldlight sensitivity threshold (FST) test. Subjects also consented
`separately to participate in an {MRI study.
`As with the initial injection, the area targeted in the readministra-
`tion was selected onthe basis ofthe results ofclinical evaluations and
`retinal imaging studies indicating that the tissue in that region had
`
`sufficient numbers of viable retinal cells. Although the subjects had
`received different doses and volumes of AAV2-hRPE65v2 intheir ini-
`
`they all received 1.5 « 10! vg in 300 pl for the
`tial administration,
`readministration study in their previously uninjected (second) eye
`(Fig. LA and Table 1). This was the same dose/volume that 46-year-old
`patient CH12 had received initially. The other two subjects (NPOT and
`CH1i, 29 and 27 years, respectively) had previously received lower
`doses (1.5 x 10" and 4.8 x 10" vg, respectivdy) in a volume of 150 pl
`(Table 1). Post-injection safety, retinal/visual function, and {MRI imag-
`ing studies were carried out seriallyat preseribed FO timePoints through
`the latest evaluation time point, FO day 180 (FOd180} (table $1).
`
`Safety of subretinal readministration
`There were no surgical complications resulting from vector readminis-
`tration. Vector was delivered to the superotemporal retina, including
`the macular region superior to the fovea, in all three individuals (Table
`1, Fig. 1, and Supplementary Methods). AHhoughthe regions ofthe
`retina that were targeted in the initially injected eve and the FO eye were
`similar, they were not entirely; symmetrical exxcept for patient CH12.
`The central retina of CH12 was scarred, and thus, the superior portions
`of the macula andretina were targeted. CH11’s second eye injection was
`slightly superior to the fovea, whereas the first injection encompassed
`the fovea; NPOI’s second eye injection occupied the superior portion of
`the macula, whereas her first injection was superotenyporal to the mac-
`ala (16, 18). AAVreadministration was well tolerated, and there was no
`inflammation in either eye of the subjects observed by clinical exam at
`any of the post-readministration time points (Fig. 1).
`There were no serious adverse events related to vector readminis-
`tration in anyof the subjects. Adverse events included surface irritation
`of the eye between POd30 and FOd60 (CH12), a sprained ankle in week
`4 (CHILD, and a headache on FOd2 (NPOI). All were deemed minor.
`Similar to previous resnits (78), blood and tear samples were posi-
`tive at lowlevels for vector DNA sequences at early post-injection time
`points (table $2). Some of the polymerase chain reaction (PCR) results
`were nonquantitative. All samples were negative after POd3. There was
`no clearrelationship between leakage of vector into the blood and im-
`roume responses (Tables 2 and 3). There were no significant cofectable
`T cell responses to either vector or transgene product (Table 2}. Two
`subjects iin this study had a transient positive eonymne.inked immunc-
`spot (ELISpot) result at a single timepoint(CHI, week 6, for AAV?
`and RPES5; NPOI, week 5, for RPE65). In both -instances, the finding
`was isolated and was not confirmed.in any other peripheral blood
`mononuclear cell (PBMC) samples collected subsequently from these
`
`
`
`Table 1. Subject enrolment characteristics and injection details.
`n LogMAR (log of the minimumangle of res-
`Visual acuity is expressed |
`olution), Higher values
`Subjects are listed in the order that they were enralled in the FO
`indicate poorer vision (see Supplementary
`conservative LogMAR
`Methocts}). Hanct motion vision was assigned a
`study. Eye #1, retina that was initially injected; Eye #2, retina that
`of 2.6.
`received the FO injection. All subjects were followed through FOd180.
`
`
`Patient ID
`
`.
`Age at
`readministration
`
`Sex
`
`Follow-up
`after initial
`injection (years)
`y
`4
`
`AAV2-hRPEGSv2 dose
`(vglivolume (al)
`Eye #1
`Eye 42
`
`Visual acuity
`i
`ipre/post)
`Eye #1
`Eye 42
`
`RPEGS mutation
`
`CH12
`
`CHT
`
`NPOI
`
`46
`
`27
`
`29
`
`F
`
`F
`
`F
`
`2
`
`2.3
`
`3.7
`
`5 x 1011/3206
`thigh/high)
`4.8 x 10'°/150
`(mediumsjiow)
`1.5 x 101/150
`(ow/low)
`
`15x 107/300
`{high/high)
`1.5% 10'1/300
`({high/high)
`1.5 x 10'1/300
`{high/high)
`
`2.6/2.16
`
`2.6/2.0
`
`K303X/AN431C
`
` 0.76/0.77
`
`—0.64/0.58
`
`VA73D/V473D
`
`1.5/1.6
`
`1.83/16
`
`E1OZK/E102K
`
`wwwSele 8 February 2012
`
`Vol 4 Issue 120 120ra15
`
`2
`
`ifOHPaPBAIUMSG4 Pephe.G10E‘O}Jaqutss8gUOWSSSLNIadaad2
`
`
`
`
`
`

`

`WOHpapRajuMeg
`
`
`G10E‘OLJaGItes3gUOMSASLNIdselye
`
`"3
`
`e=
`a
`=
`=
`a
`Bo
`=
`a
`:
`§
`te
`Na
`&
`"
`
`=
`=
`S ga
`&
`
`Pre
`
`Visual Field
`
` A
`
`Past
`
`
`iifi
`GME he3
`
`
`
`
`SOSANSS
`weeAR
`ogee.2
`
`
`
`
`
`
`tLhTe
`N
`*
`3
`sf
`BN
`x
`x
`eR ght
`as ES
`X gs
`VY gy
`ET SIS
`Ve RYN
`& MBER EN
`X SS LS
`
`XN SS ES 3
`XN SS ES §
`SARS
`Nee x
`SoS ak
`
`PIPEDPCOOERELLISEDIE
`
`
`
`
`ennnand,
`
`CNTs
`
`OH
`
`NPG
`
`(A) images of fundus photos compare
`Fig. 1.
`the baseline (Pre) and d60 (“Past’) appearance
`and the predicted pre- anc past-readministration
`visual field. There is extensive disease at base-
`line, with retinal pigment epithelial disturbance
`and geographical atrophy in the macula in pa-
`thent CH12. Arrowheadsindicate the lower border
`
`Swi
`
`GH34 & aot BOB
`& Ti,
`s sak x
`sg
`
`a RS
`&
`
`of the subretinal injection site, which was supra-
`temparal and included the superior aspect af
`the macula in all three subjects. The lower border
`of the bieb was closer to the superior vascular
`arcade in CH12, whereas the lower borders for pa-
` 3 aes tients CH11 and NPO1 were closer to the fovea. On
`; é ‘ “
`the far right are the pre- and postyeadministration
`x A ation
`visual fields, The predicted visual field changes
`PBSToengs
`based on the injection sites (and assurning a
`aie
`SH
`480
`SSG
`Serenahealthy retina) were similar for the three sub-
`the:
`Fine foses
`jects (yellow shaded areas). Gray shaded areas
`denote scotomas (spots in the visual fleld in
`which vision is absent or decreased) that were altered in location at each different FO exam (only baseline scotoermas are shown). (B) Fullfield sensitivity
`threshold testing shows an increase in retinal light sensitivity (y axis shows sensitivity thresholds) in the left eyes of NPO1 and CH11 by d30 persisting
`through the latest time point (780), but no change in sensitivity of the previously Injected eye for the three patients. There was no change in FST test
`results for either eye of patient CH12. (C) improved PLR in the second eye to receive an injection of AAV2-hRPE65v2. Average pre-veadministration PLR
`amplitudes of constriction are compared with those of post-readministration amplitudes (FOd30 to FOd180). PLR amplitudes were measured after iHu-
`mination with light at 10 lux (CH12) or 0.4 tux (CH11 and NPO1). *P = 0.08; **P = 0.009; ***P = 0.01.
`
`Apeacsdesedrteoeabete:
`fpececsteves,
`yageredececcselly
`
` we
`
` SASSSSNPSTEASTSSSASSSPSSSSNS
`
`RPM
`
`
` Servaitaity(Ht
`
`
`a, Gtrreescbonsetsassededt.
`
`3g EG
`
`~4ae
`Fkoe Ways}
`
`jects. Additionally, higher than normal background [>50 spot-
`
`forming units (SFUs) per 10° PBMCs plated in the assay] may have
`influenced the readout of the ELISpot, making the relevance of these
`findings unclear. Neutralizing antibody (NAb) responses to AAV2 and
`RPE6S protein remained at or close to baseline in the postoperative pe-
`riod in each subject (Table 2}. The rninor variations were mast likely
`due to the variability of the assay used to measure NAb. By comparison,
`NAbafter the systemic administration of an AAV2 vector in humans
`increased by several logs (4). In summary, readministration of AAV2-
`hRPEGSv2 to the contralateral eye appeared safe based on both clinical
`examination and imarmunological response.
`
`Readministration and retinal/visual function
`Each subject reported improvements in vision in the second (FO) eye
`extending over the entire period of observation beginning as early as
`FOd14. Testing revealed a trend toward improvementin visual acuity
`ofthe second eye in all three subjects, with the highest level of improve-
`
`ment in CH12. This patient also showed a trend toward improvernent
`in the initially injected eye (Table 1). There was no change in the visual
`acuity of the previously injected eye of patients CH11 and NPOL. There
`was a trend in improvement of the visual field correlating with the area
`of retina injected (Fig. 1A), although there was a high degree of intra-
`subject and intervisit variability in these subjects with low vision and
`nystagmus (involuntary, oscillating movements of the eyes}. For CH12,
`the pre- and postvisual fields were limited to a very small central island.
`
`Por CH11, the outer border of the FOd90 post-readministration visual
`fields was expanded compared to the FO baseline and FOd30 visual
`fields. For NPOL, the visual fields showed expansion at FOd45 and
`POd90 compared io baseline (Fig. 1A). There wasalso a trend regarding
`a decrease in the arnplitude of nystagrous in the initially injected eye of
`all three subjects and in the newlyinjected eve of CH11 and NPO1 (table
`55). Two of
`the subjects (CH12 and NPO1) showed reduced frequency
`ofnystagrous, whereas CH11 showedincreased frequency of nystagmus
`in both eyes after reacministration (table S5).
`
`wwwSelencelys
`fedicins.org
`
`8 February 2012
`
`Vol 4 Issue 120 120rai5
`
`3
`
`

`

`mreaes KA OTise es
`SMCaRe ARTICLES
`RESEARCH ANTECLE
`
`Table 2. Analysis of anti-AAV2 and anti-RPE65 Nab and responses over
`time after initial injection (bold) and after readministration. The exact time
`points evaluated differed for the initial and the FO study (table $1). There
`were no detectable anti-RPE65 Nabs detected after the initial injection (78).
`However, these data are not included in Table 2 because the assay was
`modified for the FO study measurements. Results are indicated as re-
`ciprocal dilutions of serum samples (see Supplementary Methods). Anti-
`AAV2 titers after the first injection were previously reported (18} and are
`
`shown here for comparison with the FO titers. The titers remained low
`throughout the course of the study, with a minor increase at week 8 for
`CHi2 (italicized) followed by a return to baseline. High FO baseline NAbs
`directed against RPEGS protein were detectabie in subjects CH12 and
`CH11. The positivity may have been due to cross-reaction with another
`RPE6S5-like protein or that the subject may produce a dysfunctional but im-
`munologically detectable protein. The positive responses detected early on
`decreased slightly over time. NA, sample not available.
`
`Subject iD
`CHI2
`
`Antibody
`assay
`AAV2
`RPE6S
`
`Baseline/FO
`baseline
`Neat-1:3.16/1:1
`+000
`
`FOd?
`1
`1000
`
`d28/FOd28
`Neat-2:3.16/1:1
`1600
`
`FOd68
`13.76-1:10
`1000
`
`dso
`Neat-1:3.16
`
`dtge/
`FOdis6
`Ww
`100
`
`d365
`Neat-1:2.16
`
`
`
`
`
`CHT 223.16-E59G/1:3.16-1:1001:10AAV2 453.96~92910/1:3.16-1:10 1:3.16-1:16 223.16-1:98 Ww 2:3, 16-1: 18
`
`
`RPE6S
`+000
`1000
`100
`100
`100
`
`
`
`
`
`ifOHPaPBAIUMSG
` ¢t
`
`oo
`al
`mi
`
`ua=4o
`
`O
`oO
`
`mw
`
`oA 3O
`
`o
`
`x&2&==
`
`> 2g
`
`S 2o
`
`a
`
`NPOt
`
`AAV2
`RPE6S
`
`<4:3.96/9:3.16-1:19
`+00
`
`1
`<100
`
`<4:3.96/1:1
`<100
`
`1
`<100
`
`<453.16
`
`T4-1:3.16
`NA
`
`2:3.76-118
`
`Table 3. Analysis of T cell responses perforrned by IFN~y ELISpot after ini-
`tial injection (cold) and after readninistration. The time points for study are
`described in table S1. Most of the samples tested for T cell responses to the
`AAV capsid or the RPE&65 transgene product were negative throughout the
`initial (78) and FO studies. A few samples tested positive in the assay (for
`example, CH12, FO week 6): however, these samples were negative the
`following week, suggesting either that the positive readings were false pos-
`
`itives or that there was weak or vansient T cell activation. Thus, there were
`no cellmediated T cell responses detectable in peripheral blood, a result in
`agreement with the lack of local inflammation. Pos, positive (>50 SFUs per
`rnillion cells plated) and at least threefold the medium-only control Neg,
`negative (<50 SFUs per million cells plated) or less than threefold the
`medium-only control: Bkg, high background/not interpretable (medium
`control >100 SFUs per million cells plated).
`
`Subject
`
`Antigen
`
` dO/FOdO week ;
`
`Week 2/FO
`week 2
`
`week 3
`
`neckA week 5 week & week 7 week &
`
`aSo/FOdSO
`
`CHI2
`
`CHT
`
`NPOt
`
`AAV
`
`RPE65
`
`AAV
`
`RPE65
`
`AAV
`
`RPE65
`
`Neg/Neg
`
`Neg/Neg
`
`Neg/Bkg
`
`Neg/Bkg
`
`Neg/Neg
`
`Neg/Neg
`
`Neg
`
`Neg
`
`Bkg
`
`Bkg
`
`Neg
`
`Neg
`
`Neg/Neg
`
`Neg/Neg
`
`Neg/Bkg
`
`Neg/Bkg
`
`Neg/Bkg
`
`Neg/Bkg
`
`Neg
`
`Neg
`
`Bkg
`
`Bkg
`
`Neg
`
`Neg
`
`*Poorviability of cells.
`
`tPositive result likely due to high background reactivity.
`
`Neg/Neg
`
`Neg/Neg
`Neg! /Bka
`Neg! /Bka
`Neg/Bkg
`Neg/Bkg
`
`Neg
`
`Neg
`Bkg
`Bkg
`Neg
`Pos!
`
`Neg
`
`Neg
`Pos!
`Pos!
`Neg
`Neg
`
`Neg
`
`Neg
`Neg
`Neg
`Bkg
`Bkg
`
`Neg
`
`Neg
`Neg
`Neg
`Neg
`Neg
`
`Neg*/Neg
`
`Neg*/Neg
`Neg/Neg
`Neg/Neg
`Neg/Bkg
`Neg/Bkg
`
`The most significant improvernents pertained to light sensitivity.
`Pull-field light sensitivity, a subjective test oflight perception, reveated
`sustained improvement in both white and chromatic (blue) light sen-
`silivity in two of the three subjects (CH11 and NPO1; Fig. 1B). One of
`these subjects (NPO1) also showed increased sensitivity to red stirnuli.
`‘he initially injected eyes retained their baseline white and blue light
`sensitivity with the exception of CH11, in whose initially injected eye
`there was diminished blue (but not white) light sensitivity after in-
`jection. Thesignificance ofthis isolated finding is unknown. Similarly,
`there were fluctuations in sensitivity in the initially injected eyes of
`OHLL and NPG] between baseline and FOd30, but levels eventually
`returned to baseline.
`Increases in ght sensitivity for the newly injected eyes were also
`detected with pupillometry. The PLR test provides objective data relat-
`ing to retinal function and the integrity of a major component of the
`retinal/central nervous system circuitry. We previously demonstrated
`that after unilateral injection of AAV2-hRPE65v2, the injected eye
`
`showed an iraproved PLR, whereas the noninjected eye rernained de-
`fective (16, 18, 19). Here, we show that there is an increased amplitude
`ofconstriction after readrninistration in each of the three FO eyes (Fig.
`1C). There were minimal changes in the amplitude of constriction of
`the initially injected eye after readministration at this sarne level of
`Huminance. Using pupilometry, we also show that in all three sub-
`jects after readrminisiration, the second eye gains responses (fig. $1).
`Further, in at least two ofthe subjects, CH12 and CHI, the initially
`
`injected eye retains its PLRs at the previous threshold sensitivity. The
`net result was that with threshold or subthreshold dhamination,
`the
`PLR waveformchanged from one suggesting arelative afferent pupillary
`defect (APD; where the initially injected eye had a robust response,
`whereas ihe uninjected eye did nat) to one that was more symmetrical
`for theleft and right eyes (fg. $1}. Although amelioration of the rAPD
`was apparent as early as FOd14, it can take months for patterns to
`stabilize and for symmetry to develop between the left and the right
`eyes. Additional follow-up testing will be necessary in these and other
`
`wwwSelencelvransiatonalMeadicing.o
`
`Vol 4 Issue 120 120ra15
`
`4
`
`

`

` TAL aaah
`
`‘
`
`:
`oy j
`
`‘
`re;
`
`subjects to determine the long-termeffects of the intervention on the
`pupillary responses of both eyes.
`The ability of the subjects to accurately navigate a standardized
`course was also evaluated (16, 18). At and before the FO baseline,
`none ofthe subjects had been able to successtully negotiate an obstacle
`course using either eye. After readministration, both NPOL and CH11
`avoided collisions with objects using their left, PO-injected eyes even
`in dim (10 fax) light for CH11 (P = 0.002 and 0.015, respectively;
`movies $1 to S4) and down to 5 tax for NPO1 (P = 0.005). Improve-
`ments in navigation were noted within 1 month after injection and
`persisted throughout the course of the study. There were no improve-
`ments in navigation using the initially injected eye.
`
`Readministration and cortical respanses
`{MRI analyses were performed with the general linear model and the
`contrast of active blocks (checkerboard stimuli) minus the rest blocks
`(black screen) (fig. S2) using the BrainVoyager QX software (22). To
`
`account for variability in the disease stage ammong subjects, we analyzed
`fMRIindividually for each participant (20) (and not groupedas in most
`fMRI analyses). A single-subject analysis approach was especially suit-
`
`able based onthefact that thethree subjects differed by age and disease
`progression andthus differed in the areaof the retina in which there was
`evidence of sufficient (albeit unhealthy) retinal cells. This approachalso
`makes the correlation of fMRIresults and clinical outcomes possible for
`eachindividual. All analyses were carried out to obtain significant results
`at high statistical thresholds that were correctedfor false detection of any
`activation due to multiple-cornparisontype I errors (23); the thresholds
`were lowered if no activation was detected. At a lowerstatistical thresb-
`old, there was frontal activation responsible for eye movement (frontal
`eye fields), anterior cingulate (decision-making for button press), and
`premotor and sensory motor cortex (for button press).
`
`fMRI results for newly treated eyes
`ARE after gene therapy readministration showed significant cortical
`activation in and around the visual cortex for all three LCA2 subjects
`for full-field contrast-reversing (8 Hz) checkerboard stimuli at high
`and riediarn contrasts (Figs. 2 to 4). Presentation of ihe same stimull
`at baseline, before readministration, did not result in significant cor-
`tical activation for either the high- or the medium-contrast stirnulus.
`‘The results for each subject are as follows.
`
`CH12’s untreated eye before readministration was unresponsive to
`the high- and medium-contrast stimuli (Fig. 2, A and B) even at liberal
`statistical threshold levels. Significant bilateral cortical responses to the
`high-contrast
`sistimulus were observed: false discovery rate (fdr) was
`<5% with a corrected Pvalue((P.} of <0.002 and continuously connected
`area {cca} of S100 many’; no response fo mediumcontrast was recorded
`at FOd30(Fig. 2, C and D, respectively}. Even though her FO baseline
`and posttreatment visual fields were limited to a very small central area
`{Fig. 1), CH12’s cortical responses to the high-contrast stimulus mark-
`edlyincreased at FOd90 (Fig. 2, E and F}, especialy for the high-contrast
`stinwulus (fdr < 5%, P. < 0.005, cca = 1000 mm”). The mediur-contrast
`stironlus showedunilateral but siggnificant (ide < 5%, P< 0.0002, cca =
`25 mm’) cortical activation.
`CH11 showed nocortical activation, regardless of visual stimulus
`presented to her untreated (left) eye at FO baseline (Fig. 3, A and B).
`However, widespread bilateral activation was observed for the (MRI
`obtained on FOd30in response to the high- and medhur-contraststimu-
`li (fdr < 5%, P. < 0.003, cca > 1000 mm’) (Fig. 3, C and D), and the areas
`
`Gasaiine
`
`
`at baseline, FOd30, and FOd90. (A and B)
`Fig. 2. Subject CH12 fMRI results
`Subject CH12 showed no cortical activation at baseline for high- and medium-
`contrast stimull. (C and B) Ar FOd30, significant bilateral cortical activations
`were observed in response to the high-contrast stimulus (C), whereas no
`response was recorded for the medium-contrast stimulus (D). (E and F) At
`FOd90, CH12’s cortical responses to the same stimuli markedly increased
`especially for the high-contrast stimulus. Smaller clusters of activations are
`observed in response to medium-contrast stirnulus at FOd90 (F).
`
` WOHpapRajuMeg
`
`
`G10E‘OLJaGItes3gUOMSASLNIdselye
`
`of activation increased by FOd90 (Fig. 3, E and P). At FOd90 (Fig. 3B),
`there was greater bilateral cortical activation for the high-contrast stim-
`ulus (fdr < 5%, P. < 6.003, cca 2 1060 mm"). Markedactivation was also
`present in response to the t nedium-contrast stimulus (fdr < 5%, P. <
`0.003, cca = 1000 mm”) (Fig. 3F). As depicted in Fig. 3, CH11’s FO
`visual activations were symmetrically distributed in both hemispheres
`as well as in the upper and fower banks ofthe calcarine fissure, compa-
`rable to a pattern predicted from her visual field distribution and the
`
`loceation. ofthe subretinal injection (Fig. 1), giventhat the cells in the
`injected region were viable.
`‘Similar|te CHL and CH12, NPO1 did notpresent with anyactiva-
`tion in response to the high- or medium-contrast stimuli for her un-
`treated eye at FO baseline (Fig. 4, A and B} At FOd45, there was a
`response to the high-contrast stimulus (Pig. 4C; fdr < 5%, P. < 0.001,
`cca > 50 mm’), but not to the medium-contrast stirmalus (Fig. 4D). The
`clusters of activation were bilaterally distributed and mainly located in
`the lateral and basal areas of the visual cortex, generallyreflective of a
`at
`pattern predicted by the FO visual fields (Fig. 1). At FOd90, NPOL
`showed increased bilateral activation in response to both the high-
`contrast (fdr < 5%, P. < 6.0003, cca = 100 mmand the medium-
`contrast (fdr < 5%, P. < 0.001, cca = 25 mm*)stimuli as depicted in
`Fig. 4, E and F, respectively.
`Quabitative [MRI teroporal changes for the PO studies of al} three
`subjects are summarized in table $3. Results show that cortical re-
`sponses increased in all subjects from baseline to FOd30 and continued
`to FOd90, Quantification of the fMRI results (areas of activation, mm*)
`for each hemisphere and total visual cortex for the FO studies are
`presented in table S4. Results showthat the areas of visual cortex ac-
`tivation after visual stimulation increased in all three subjects through
`POd90 (P < 0.0001, table $4). Steady increases in total cortical activa-
`tion areas through FOd90 forall three subjects agreed with the increased
`
`WWSeieneel ya
`
`ins.org
`
`8 February 2012
`
`Vol 4 Issue 120 120rai5
`
`5
`
`

`

` TAL aaah
`
`‘
`
`oy j
`
`re;
`
` High
`
`corirast
`
`Mediumcontrast
`
`
`
`Fig. 3. Subject CH11 fMRI results at baseline, FOd36, and FOd90, (A and
`8) Subject CH11 showed no baseline cortical activation to the high- or
`medium-contrast checkerboard stimull. (€ and B) Highly significant and
`widespread bilateral activation at FOd30 in response ta both high- and
`medium-contrast stimuli, respectively. (E and F) A more marked increase
`in cortical activation was present at FOd90 for high-contrast (F) and medium-
`contrast (F) stimull,
`
`light sensitivity measured with PLR testing and, for twoof the sub-
`jects, with FST testing, in the same timeframe (fig. Si and Fig. 1C). This
`mayreflect increasing expression of the RPE6S transgene over this time
`period. The largestrelative gains were observed in CHI2 and NPOL, the
`oldest of the three subjects. All subjects presented with greater bilateral
`activation at FOd90, This is not surprising because the subretinal in-
`jections spanned the midline ofthe posterior pole of the eye and thus
`should affect both hernispheres. There was good correlation between
`the {MRE findings and theresults of retinal and visual functiontesting.
`In particular, the incrernental increase in total cortical activation areas
`through POd90 correlated with average postsurgical pupil constriction
`arnplitudes (P < 0.049).
`In summary, results from fMRI showedan increase in cortical ac-
`tivation after readministration of gene therapy, and the pattern of vi-
`
`anal cortex activation roughly correlated with the location of injection
`and visual field distribution. Termporal increases in cortical activation
`also generally correlated in time and magnitude with those that were
`
`
`measured using psychophysical testing.
`
`MBI results far previously treated eye
`Tn addition to the newly treated eye, {MRI was also performed on the
`eve that had been initially injected at least 1.7 years earlier (see Table 1).
`‘This experiment was carried out to evaluate the functionality of the
`contralateral eye and to evaluate any potential toxicity associated with
`readministration of gene therapy. {MRI for the contralateral eye was
`carried out at FO baseline and FOd90.
`As shownin Fig. 5, {MRI results at FO baseline for CH12 showed
`bilateral activation, distributed more extensively in the lateral aspects
`of the visual cortex, in response to high-contrast stirauli (fdr < 5%, P. <
`O.OL, cca > 25 mm?) and at an uncorrected statistical level (P < G.01,
`cca > 25 mm’) for medium-contrast stimuli. CH11 showed bilateral
`
`
`
`Basaline
`
`Day 48
`
`Bay 80
`
`
`ighcontrast
` WOHpapRajuMeg
`
`Mediumcontrast
`
`
`G10E‘OLJaGItes3gUOMSASLNIdselye
`
`Fig. 4 Subject NPO1 FMBI results at baseline, FOd4S, and FOd90. (A and
`8) Subject NPO1 showed no visual activation at baseline. (C and BD) At
`FOd45, although significant cortical resconses for the high-contrast stim-
`ulus were recorded (C}, no response was observed for the medium-contrast
`stimulus (D). (E and F)
`At FOd90, NPG1 showed significant activation for
`high-contrast (E}) and medium-contrast {F) stimuli. Areas of activation at
`FOde0 were distributed in closer proximity to the primary visual cortex
`compared to FOd45 fMRI results [compare (E) and (Ci).
`
`activation for high-comtrast stimuli (fdr < 5%, P.< 0.01, cca > 100 mn}
`and no activation for medium-contrast stinvali. The {MRI results for
`NPOL were observed at an uncorrected fdr statistical level for high-
`contrast stimuli (P < 0.01, cca > 25 mm‘), with no activation detected
`for medium-contrast stimuli.
`The {MRI results for the initially injected eyes al FOd90are presented
`in Fig. 6. AH three subjects demonstrated bilateral activation in re-
`sponse to the bigh- and medium-cont