Safety of Recombinant Adeno-Associated Virus Type 2–RPE65 Vector Delivered by Ocular Subretinal Injection

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doi:10.1016/j.ymthe.2006.03.005

Safety of Recombinant Adeno-Associated Virus Type 2–RPE65 Vector Delivered by Ocular Subretinal Injection Samuel G. Jacobson,1,* Gregory M. Acland,2 Gustavo D. Aguirre,3 Tomas S. Aleman,1 Sharon B. Schwartz,1 Artur V. Cideciyan,1 Caroline J. Zeiss,4 Andras M. Komaromy,3 Shalesh Kaushal,5 Alejandro J. Roman,1 Elizabeth A. M. Windsor,1 Alexander Sumaroka,1 Susan E. Pearce-Kelling,2 Thomas J. Conlon,6 Vincent A. Chiodo,5 Sanford L. Boye,5 Terence R. Flotte,6 Albert M. Maguire,1 Jean Bennett,1 and William W. Hauswirth5,6 1

Scheie Eye Institute, Department of Ophthalmology, University of Pennsylvania, Philadelphia, PA 19104, USA 2 James A. Baker Institute for Animal Health, Cornell University, Ithaca, NY 14853, USA Section of Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA 4 Section of Comparative Medicine, Yale University School of Medicine, New Haven, CT 06520, USA 5 Department of Ophthalmology, University of Florida, Gainesville, FL 32610, USA 6 Powell Gene Therapy Center, Genetics Institute, Department of Pediatrics, Department of Pathology, and Department of Pharmaceutics, University of Florida, Gainesville, FL 32610, USA 3

*To whom correspondence and reprint requests should be addressed. Fax: +1 215 662 9938. E-mail: [email protected].

Available online 27 April 2006

AAV2 delivery of the RPE65 gene to the retina of blind RPE65-deficient animals restores vision. This strategy is being considered for human trials in RPE65-associated Leber congenital amaurosis (LCA), but toxicity and dose efficacy have not been defined. We studied ocular delivery of AAV-2/2.RPE65 in RPE65-mutant dogs. There was no systemic toxicity. Ocular examinations showed mild or moderate inflammation that resolved over 3 months. Retinal histopathology indicated that traumatic lesions from the injection were common, but thinning within the injection region occurred only at the two highest vector doses. Biodistribution studies at 3 months postinjection showed no vector in optic nerve or visual centers in the brain and only isolated non-dose-related detection in other organs. We also performed biodistribution studies in normal rats at about 2 weeks and 2 months postinjection and vector was not widespread outside the injected eye. Dose–response results in RPE65-mutant dogs indicated that the highest 1.5-log unit range of vector doses proved efficacious. The efficacy and toxicity limits defined in this study lead to suggestions for the design of a subretinal AAV-2/2.RPE65 human trial of RPE65associated LCA. Key Words: adeno-associated virus, dog, gene therapy, Leber congenital amaurosis, retina, RPE65

INTRODUCTION Proof-of-concept studies in canine and murine animal models of RPE65 (retinal pigment epithelium-specific protein 65 kDa)-deficient Leber congenital amaurosis (LCA), an autosomal recessive human retinal disorder [1,2], demonstrate restoration of vision to blind eyes by subretinal delivery of RPE65-expressing recombinant adeno-associated virus (rAAV)-based vector [3–8]. Efficacy of subretinal rAAV.RPE65 in the RPE65-mutant dog has been demonstrated in relatively short-term studies and

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persistence of the visual restoration for years has been documented [3,4,8]. These preclinical studies are sufficiently promising to warrant consideration of human clinical gene transfer for this otherwise incurable form of genetic blindness [9–11]. Increasing information has become available about the safety profile of intraocular delivery of different rAAV serotypes [12,13], but specific toxicology and biodistribution studies of subretinal rAAV.RPE65 have not been published. The present investigations in large and small

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animals report vector dose efficacy and vector-related toxicity and biodistribution after subretinal delivery of rAAV-2/2.RPE65.

RESULTS We assessed the safety of rAAV-2/2.RPE65 in RPE65mutant dogs and normal rats. Schematics of the vectors used (Figs. 1A and 1B) show that they contain two AAV2 inverted terminal repeats that flank a regulatory element composed of the cytomegalovirus (CMV) immediate early enhancer, chicken h-actin promoter with first intron/exon junction, hybrid chicken h-actin and rabbit h-globin intron/exon junction followed by a human RPE65 cDNA, and the SV40 polyadenylation signal. The vectors, denoted CBo (Fig. 1A) and CBSB (Fig. 1B), differ by 152 bp at the 5V end of the CMV immediate early enhancer. We used vector CBo in 15 dogs and the longer term rat study. We used vector CBSB in 3 dogs and the short-term rat study (Table 1; Supplementary Table 1). The vector plasmid DNA from which all vectors were made has a kanamycin resistance gene in the plasmid backbone for selection, which is not present in the final vectors. Canine Safety Studies We performed a 3-month safety study in 15 RPE65mutant dogs: 2 dogs with bilateral ocular injections of vehicle control and 13 dogs with bilateral injections of rAAV-2/2.RPE65 at different dose levels (Table 1). The dogs were between 2.7 and 7.4 months of age at the time of ocular surgery. The retinal site and extent of all injections, based on drawings after surgery, are shown for eyes that received vehicle control (Fig. 2A) and vector (Fig. 2B). All dogs remained clinically healthy for the duration of the experiments (see supplementary material for in-life observations and clinical pathology). We studied the potential for a humoral immunologic response to AAV2 capsids after subretinal injection in a

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subset of six dogs (D5, D9–D12, and D15), representing relative vector doses ranging from 0.001 to 3 (Table 1). We assayed sera collected before and 5 weeks after injection by ELISA. Specifically, we allowed sera from these dogs and a canine anti-AAV2 Ig standard to incubate in the presence of AAV2 capsids and detected absorbance. Sample dilutions ranged from 1:10 to 1:10,000, which allowed us to observe potential inhibition due to the prozone effect. We set the limit of detection at 1.9 mU/ml based on the linear range of the standard curve. Pretreatment levels were near or below the limit of detection and there was no observable increase (all showed a decrease) in antibody response 5 weeks after vector treatment. In vivo examination results of preretinal ocular structures at four times postinjection are summarized in Fig. 3. We graded abnormalities at three levels of severity. Among the notable findings at 1 week postinjection were mild or moderate conjunctival reaction (3 of 4 vehicle-control eyes; 12 of 26 vector-injected eyes) and vitreous cellularity (1 of 4 vehicle-control eyes; 8 of 26 vector-injected eyes). The conjunctiva cleared in almost all animals by 3 months. Mild abnormalities at 3 months persisted in the vitreous of 5 eyes; 1 of the eyes had a vitreous hemorrhage from ocular surgery (D6, left eye (LE)). We observed unilateral posterior subcapsular lens opacities in two dogs. We noted small anterior lens opacities in 4/30 eyes (not plotted in Fig. 3) following accidental trauma during paracentesis. Intraocular pressure at all times did not differ from normal [14,15]. The most common ophthalmoscopic abnormality at 3 months postinjection (25 of 30 eyes) was a linear or curvilinear pigmented lesion in the tapetal retina at the presumed site of the retinotomy. Five eyes had other changes. D9 (LE) had an intratapetal injection and there were marked changes in tapetal reflectivity in the injection area. D12, with an inferior retinotomy in the LE, showed depigmentation in the injection region. FIG. 1. Design of the rAAV-2/2.RPE65 vectors in this study. (A) AAV2-CBo-hRPE65, 4070 bp, and (B) AAV2-CBSB-hRPE65, 3921 bp, differ by 152 bp at the 5V end of the CMV immediate early enhancer. ITR, AAV2 inverted terminal repeats; CMV ie enhancer, cytomegalovirus immediate early enhancer; h-actin, chicken h-actin promoter; Exon1, chicken h-actin exon 1; Intron, hybrid chicken h-actin and rabbit h-globin intron; Exon2, rabbit h-globin exon 3; hRPE65, human RPE65 cDNA; SV40 poly(A), SV40 polyadenylation signal.

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TABLE 1: Characteristics of RPE65-mutant dogs and ocular rAAV doses Animal Vehicle injectedb D1 (BR246)

Gender M

Eye

Dose (relative)a

R 0 L 0 D2 (EMB31) F R 0 L 0 Vector injected (AAV2/2-CBo-hRPE65)b D3 (BR244) M Rc 0.0001X L 0.0001X D4 (BR239) F R 0.0001X L 0.0001X D5 (BR256) F Rc 0.001X L 0.001X D6 (EMB33) F R 0.001X L 0.001X D7 (BR251) F R 0.01X L 0.01X D8 (EMB28) M Rd 0.01X L 0.01X D9 (BR257) F R 0.03X Le 0.03X D10 (BR263) F R 0.1X L 0.1X D11 (BR266) F R 0.3X L 0.3X D12 (BR264) F Rf 0.3X L 0.3X D13 (BR248) M R 1X Lc 1X D14 (BR235) M R 1X L 1X D15 (BR265) F R 3X L 3X Vector injected (AAV2/2-CBSB-hRPE65)g D16 (EMB62) F R 0.1X 0.1X Lc D17 (EMB59) F R 0.3X L 0.3X D18 (BR303) F R 1X L 1X

Vector genomes delivered (1010)a 0 0 0 0 0.015 0.015 0.015 0.010 0.15 0.15 0.15 0.15 1.5 1.5 1.5 1.5 4.5 4.5 15 15 45 45 45 30 150 150 150 150 450 450 15 15 45 45 150 150

section of brain; the significance of this observation is unknown. Ocular histopathology revealed a spectrum of lesions (Figs. 4A–4I). Some lesions were attributable to retinal surgery, while others were related either to the underlying disease process or possibly to the vector. Traumatic retinal lesions from the surgery ranged from focal retinal perforation with disorganization of the outer nuclear layer (ONL), to segmental retinal disruption and fragmentation with some atrophy, to complete retinal rupture. Fig. 4A shows a transretinal lesion in the superior tapetal region, site of the subretinal injection, of the RE of D7 (0.01); the lesion has displacement of photoreceptor nuclei, probably representing a needle tract. An almost full-thickness retinal perforation was present in the RE (subretinal injection zone) of D3 (0.0001) with disorganization of the ONL (Fig. 4B). A more extensive fullthickness defect was present in D1 (vehicle) in the temporal retina (injection site, RE) with the retinal edge reflected back and focal disarray of photoreceptor cells (Fig. 4C). We also noted inflammatory lesions. Small numbers of macrophages and lymphocytes were evident in the vitreous (Figs. 4C and Figs. 4D, from D1, RE, and D6, RE, respectively). Occasional perivascular lymphohistiocytic infiltrates were present in retinal vessels (Fig. 4E; D6, RE). Retinal abnormalities in the superior central region of the RE of D15 (3) were notable. This eye had an extensive retinal detachment in this region (subretinal injection site) with attendant loss of RPE and photoreceptor outer segments, accumulation of subretinal debris and macrophages, rosette formation (Fig. 4F), and vascular-associated adhesions of the retina to tapetum. Focal ONL atrophy was present. We found ONL thinning to different degrees in the superior central

R, right; L, left. a 1X, 1.0  1010vg/Al (injections were 150 Al in volume, except in D4, LE, 100 Al; D12, LE, 100 Al). b Safety and efficacy studies. c Subretinal/intravitreal. d Intravitreal. e Intratapetal. f Sub-RPE. g Efficacy studies only.

D11 (LE), D13 (LE), and D15 (right eye (RE)) showed areas around the retinotomy site suggesting retinal thinning, but the contralateral eyes were not affected in this way. Pathological examination of nonocular tissues showed no specific abnormalities. Optic nerves and chiasms were normal. D13 had a single necrotic neuron in a single

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FIG. 2. Schematics showing the sites of subretinal injections in the 18 RPE65mutant dogs of this study. Injection sites of (A) vehicle control and (B) rAAV-2/ 2.RPE65 vector are shown for right eyes (RE) and left eyes (LE).

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FIG. 3. In vivo ocular examination of 15 RPE65 mutant dogs after ocular delivery of rAAV-2/2.RPE65 vector (circles) compared to vehicle control (diamonds). Inflammatory changes (conjunctival hyperemia, chemosis, or discharge; cellularity/precipitates in anterior chamber and/or vitreous) as well as changes in transparency of ocular media (cornea/lens/vitreous) were assessed by slit-lamp biomicroscopy and indirect ophthalmoscopy. Clinical changes were graded at three levels of severity. Results from individual eyes are presented from left to right in order of increasing relative vector dose levels; data points are arbitrarily offset in the vertical direction within each dose level and ocular evaluation category. Eight vector doses (Table 1) are represented by levels of gray in the symbols (scale at bottom right).

region (subretinal injection site) of the two dogs that received 1 vector dose (D13, Fig. 4H; D14, Fig. 4I); for comparison, the same retinal location is shown for the vehicle-injected RE of D2 (Fig. 4G). D13 has not only reduced ONL but also reduced OS (left eye) length; D14 shows even greater ONL and OS reduction and pale gray material in the subretinal space. We measured thickness of the ONL along the vertical meridian (passing through the optic nerve) in the 27 eyes

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(23 vector and 4 vehicle) with subretinal injections (Table 1, Fig. 4J). We estimated the locations corresponding to the subretinal injection region (Fig. 4J, red outlines) using either in vivo fundus drawings (Figs. 2A and 2B) alone or the drawings together with RPE65 immunocytochemistry results, when positive. Visual inspection of the quantified data shows that there was gross thinning of the ONL in some eyes with 1 and 3 doses. Thinning generally coincided with but was not always limited to the region of the subretinal injection (Fig. 4J). For statistical analysis, we first produced an average control ONL thickness profile for the age-matched RPE65-mutant retina using measurements from uninjected regions of the four control eyes. Next, we derived the percentage difference from this control ONL thickness for each sample. For each eye, we averaged samples corresponding to injected and uninjected regions and plotted them, grouped by vector dose (Fig. 4K). There were no statistically significant differences between injected and uninjected regions for doses from vehicle through 0.3. Statistically significant differences (Fig. 4K, asterisks) were found for four of six eyes with 1 and 3 doses (t test; P b 0.002 adjusted for multiple comparisons performed). We studied vector biodistribution at 3 months after subretinal injection in ocular tissues and other organs of the 15 dogs (Table 2). In the optic nerve and chiasm of the two dogs receiving vehicle, there were no detectable vector sequences. We were able to analyze 22 samples of optic nerve from vector-injected eyes and 21 had no detectable vector sequences. D10 (LE, subretinal 0.1 dose) showed detectable vector sequences, but the result was not replicated on retest. Of note, among these 22 eyes were 2 with partly subretinal injection and partly intravitreal injection and 1 eye with an intravitreal injection (Table 1). Optic chiasms were negative for all vector-injected animals except D13 (1 dose), and this was a single value that did not replicate. We sampled 5 vector-injected dogs (D5, D9–11, D15) for vector sequences in regions of the visual pathway beyond the chiasm. We found no detectable vector sequences in the 15 samples analyzed (Table 2). Other organs in all 13 vector-injected dogs were negative for vector sequences, with 3 exceptions. D6 (0.001 dose) had positive results for heart and diaphragm. D4 (0.0001 dose) and D13 (1 dose) had a single unreplicated vector sequence in a mandibular node; D13 also had an unreplicated positive result in the diaphragm. The significance of these results is uncertain. Canine Efficacy Studies at Different rAAV-2/2.RPE65 Doses RPE65 deficiency in the dog leads to reduced electrophysiological responses of the retina to light [3,4,8]. We used the electroretinogram (ERG), an objective and noninvasive method to quantify retinal function [16], to

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FIG. 4. (A–I) Retinal histopathology and (J, K) outer nuclear layer (ONL) thickness analyses in 15 RPE65-mutant dogs at 3 months after intraocular delivery of vehicle or rAAV-2/2.RPE65 vector. Histological sections illustrate injection site traumatic lesions (A–C), inflammatory changes in the vitreous and retina (C–E), rosette formation within a large traumatic lesion (F), and outer retinal thinning at dose 1 (H, I) compared with vehicle (G). Note that the images in G, H, and I are from the same retinal location. Calibration bars (A, D, G) apply to entire rows of micrographs except F, which is at a higher magnification. (J) ONL thickness measurements along the vertical meridian of the eyes of the 15 dogs studied; circles and triangles within each vector-dose graph represent different animals (filled, RE; open, LE). Black lines connect individual measurements in each eye. Thicker red lines define region of subretinal injection (in this meridian). (K) Summary data comparing uninjected (black squares) with injected (red squares) retina for each eye, within each vector-dose level. Data from the same eye are connected by a vertical line. *Statistically significant difference between uninjected and injected data for that eye. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

determine the response to different doses of rAAV-2/ 2.RPE65 in the same dogs from which we gathered safety data and in three additional dogs (D16–D18; Table 1). In normal dark-adapted dogs, ERGs to increasing intensities of light stimuli become greater in amplitude and more complex with subcomponents (Fig. 5A, left). In contrast, an RPE65-mutant dog (D2, LE; vehicle injected) shows no ERG responses to these lights (Fig. 5A, middle) and this is typical of untreated dogs [3,8]. Subretinal rAAV-2/ 2.RPE65 in another RPE65-mutant dog (D13, RE; 1 dose) leads to ERGs that are normal in threshold, but subnormal in amplitude (Fig. 5A, right). Light-adapted ERGs to flickering stimuli are shown for a normal dog and for the same RPE65-mutant dogs (Fig. 5B). The eye treated with subretinal rAAV-2/2.RPE65 shows a reduced but measurable ERG with a waveform like that of the normal; no response was detectable in the vehicle-injected eye. We used two conventional stimuli to determine the relationship of ERG response to vector dose: a darkadapted white flash (waveform to highest intensity in

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Fig. 5A) and light-adapted flicker (Fig. 5B). We graphed response amplitude from all eyes with subretinal injections and compared it with data from normal dogs, untreated RPE65-mutant dogs, and the 2 vehicle-injected dogs (Fig. 5C). We considered response positive if amplitude was N3 SD from the mean amplitude of the untreated RPE65 -mutant dogs (dark-adapted stimulus mean response amplitude F SD, 2.9 F 1.3 AV; light-adapted stimulus mean response amplitude F SD, 0.5 F 0.5 AV; n = 45 eyes of 23 dogs, ages 2–11 months). There was definite vector dose dependence in the ERG data. Taking together all doses z0.1, 17 of 18 eyes (94%) responded to the darkadapted stimulus and 13 of 18 eyes (72%) to the lightadapted flicker. Specifically considering the 10 eyes treated with 0.1 (n = 4) or 0.3 (n = 6) dose, all but 1 eye (at 0.3) responded to the dark-adapted stimulus; flicker ERGs showed less responding success, with 2 eyes from each of these dose groups being no different from untreated eyes. At doses b0.1, only 1 eye (D6, LE; 0.001 dose) of 14 (7%) showed a response that was greater than those of the 4

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Optic nerve L Optic nerve R Chiasm Optic tractc Lateral geniculatec Opt. radiationc Visual cortexc Superior colliculusc Periocular L Periocular R Mandibular node L Mandibular node R Parotid node L Heart L Heart R Lung Diaphragm Liver L Liver R Pancreas Spleen Kidney L Kidney R Jejunum Gonads L Gonads R Skeletal muscle

D2 0

D3 0.0001X

D4 0.0001X

D5 0.001X

D6 0.001X

D7 0.01X

D8 0.01X

D9 0.03X

D10 0.1X

D11 0.3X

D12 0.3X

D13 1X

D14 1X

D15 3X

– – – nd nd nd nd nd – – na na – na na na – na – na na – – na – – –

– – – nd nd nd nd nd – – na – – – na – – na na – – – na na – – na

– – – nd nd nd nd nd – – na na na – – na – na na – – – – – – – –

– – – nd nd nd nd nd – – F(100) – – – na na – na na – – – na na – na –

– na – – – nd – – – – – – – – – – – – – – – – – na – – –

– – – nd nd nd nd nd – – – na na +(565) – – +(1141) – na – na – – na na – –

– – – nd nd nd nd nd – – – – – – na – na – na – – – – – – – –

na – – nd nd nd nd nd – – na na – – – – na na na – na – – – – – –

– – – – – nd na nd – na – – – – – – – na – – – – – – – – –

F(263) – – – – na – nd – – na na – – – – – na – – – – – – na – –

– na na nd – nd – – na na na – na na na na na na na – na – na – – – na

na – – nd nd nd nd nd na na na – – – – – na – – na na – – – na – –

– – F(101) nd nd nd nd nd – – na F(580) – – – – F(196) – – – – – – na – – –

– – – nd nd nd nd nd – – – na – – – – – na na – na – – – – – –

– – – – – nd – nd – – na – – – – – – – – – – – – – na – –

doi:10.1016/j.ymthe.2006.03.005

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TABLE 2: Detection of rAAV vector sequences by PCR in RPE65-mutant dog tissue samples D1a 0b

L, left; R, right; –, no PCR amplification; PCR amplification of vector sequences: +, replicated value shown is average, copy number per Ag of DNA; F, single replicate value only, copy number per Ag of DNA; na, not available (not determinable because of an unacceptable spike-in); nd, not done. a Animal. b Vector dose, relative to Table 1. c Left brain sampled only.

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FIG. 5. Retinal function after subretinal delivery of different doses of rAAV-2/2.RPE65 vector to 18 RPE65- mutant dogs. (A) Dark-adapted ERGs evoked by increasing intensities of light in RPE65-mutant dogs 2–3 months after subretinal delivery of rAAV-2/2.RPE65 (dose 1; right) compared to an age-matched vehicle-controlinjected RPE65-mutant dog (middle). ERGs from a normal dog are also shown (left). Stimulus onset is at trace onset; stimulus intensity is at the left of key traces; calibration bars, bottom right of responses. (B) Light-adapted flicker (29 Hz) ERGs elicited by white-flash stimuli in the same animals. Vertical gray bars represent stimulus onset; calibration bars, right of responses. (C) ERG amplitudes under dark- and light-adapted conditions in normal (N) dogs (squares) and in different groups of RPE65-mutant dogs: untreated (U) eyes (hexagon; error bar, mean + 3 SD), vehicle control (VC)-injected eyes (diamonds), and eyes injected with increasing dose of rAAV-2/2.RPE65 vector (circles, vector AAV2-CBo-hRPE65; circles with dots, AAV2-CBSB-hRPE65). Eight levels of vector doses are represented by levels of gray fill.

vehicle-injected eyes or untreated RPE65-mutant dogs from our earlier studies [3,8]. We performed immunocytochemical staining of RPE65 protein in retinal histological sections along the vertical meridian of RPE65-mutant dogs in the safety studies (Table 1). Immunolabeling, when present, was limited to the RPE cells, as previously demonstrated [8]. It is of interest to relate RPE65 protein staining to vector dose and ERG response. We detected no RPE65 staining in the sections from eyes with vehicle injection or vector injections V0.1 dose. Of interest, the 0.1 dose showed ERG responses. Of the four eyes with the 0.3 dose, only one showed detectable RPE65 immunostaining (D11, LE); this eye also had ERG responding. All six eyes at doses of 1 and 3 showed both RPE65 staining and ERG responding. Rat Biodistribution Studies We performed studies in normal rats to assess rAAV-2/ 2.RPE65 vector spread to distant organs after subretinal or intravitreal injection of doses approximate to those that showed ERG efficacy in the dog. We investigated a relatively acute period (10–17 days) and a longer interval (7–8 weeks). We analyzed the injected eye (with attached intraorbital optic nerve) for vector sequences and sampled 14 other tissues (Supplementary Tables 1, 2, 3).

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First, we studied vector spread after 10–17 days in 13 rats with 1 subretinal vector and, to test a worst-case situation, 6 rats with 3 intravitreal vector (Supplementary Table 2). The injected eye was positive (and replicated) for vector sequences in all rats with subretinal injections and in 4 of 6 intravitreal-injected rats; the other 2 rats in the latter group showed no detectable or replicated vector sequences in any of the other 14 tissues and are not further considered due to technical uncertainties. For the 1 subretinal injections, most of the other sampled tissues, including gonadal and brain samples, were negative for vector sequences (see supplementary tables for exceptions). For the 3 intravitreal injections, 1 animal had a replicated positive result in muscle (134 copy number per microgram of DNA). The results suggest that at short periods after subretinal or intravitreal vector delivery, there is very limited vector DNA detectable outside of the injected eye. Second, we studied vector spread after 7–8 weeks in 16 rats with different doses of subretinal vector and 4 rats with intravitreal vector (Supplementary Table 3). In the 0.25 subretinal group, four of the seven reportable values for the injected-eye optic nerve tissue showed no vector sequences (one was not determinable). One sample of four in the intravitreal group also showed no detectable vector sequences in the injected-eye optic

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nerve. All other eye optic nerve tissue yielded values greater than 100 copies/Ag of DNA. This is consistent with a progressive loss of vector sequences with time from the site of administration, and as expected, this was primarily seen with the lower subretinal doses. At the lower dose subretinal injections, most sampled organs including gonads and brain were negative. In the higher dose subretinal group, there were three positive but not replicated results. Rats with intravitreal injections showed no positive organs for vector sequences. Thus, rat biodistribution analyses at 7–8 weeks after vector administration suggest the ocularly delivered vector does not spread widely outside the injected eye.

DISCUSSION The risks of gene transfer in general and ocular gene therapy in specific have been discussed [17–20,10]. Two reports with safety data were recently published: one was concerned mainly with biodistribution [12] and the other with clinical ocular data [13]. These reports included experiments with subretinal rAAV2.CMV.gfp using serotypes 2, 4, and 5. Of relevance to the present work, five rats had subretinal rAAV-2/2 and showed detectable vector sequences in the injected retina and its optic nerve but not in brain or other organs when sampled at 1–3.5 months postinjection [12]. The clinical study used subretinal rAAV-2/2 in one normal dog. This dog had no signs of ocular inflammation or toxicity; ERGs showed no interocular asymmetry as long as 3 years after the uniocular injection [13]. Vector dose in both studies was approximately equal to the 0.1 dose of the present work. Our studies of biodistribution, safety, and efficacy of ocular-delivered rAAV-2/2.RPE65 help fill the gap between proof-of-concept [3–8] and use of this material for human gene therapy. Systemic or retina-wide toxicity was not detectable in the dogs at any vector dose of this localized retinal treatment. Localized retinal toxicity, however, was demonstrable with quantitative ocular histopathology in the dog. The data suggest there are both vectorindependent and vector-dose-dependent components to this localized toxicity. The vector-independent component was due mainly to subretinal surgery (i.e., the intentional perforation and detachment of the retina to deliver the test agent). Many previous studies of experimental retinal detachment in normal animals (reviewed in Ref. [21]) have shown that a localized retinal detachment can lead to localized photoreceptor loss and RPE changes; and treatment-induced retinal trauma from intraocular gene therapy has been documented [22]. Neural and glial remodeling is also expected to occur [21]. Studies of neurotrophic factor release after needle trauma to the retina suggest there may be repair processes occurring simultaneously [23].

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A vector-dose-dependent component of the localized toxicity occurred in some but not all eyes receiving higher doses in the dog. Four of six eyes receiving 1 or 3 vector doses showed outer retinal cell loss. A counterintuitive result was the coexistence of treatment efficacy with evidence of toxicity. In these eyes, local reduction of photoreceptor numbers due to surgical trauma, vector toxicity, and the underlying retinal degeneration phenotype may be superimposed on the efficient restoration of the visual cycle in the diseased but remaining cells. Immunocytochemical staining for RPE65 in the 1 and 3 eyes proved that the protein was produced in the RPE of treated regions of these eyes. The primary cellular site or exact mechanism of the vector toxicity is not known. The present study could not distinguish between the three likely choices: photoreceptor numbers were reduced exclusive of RPE reduction, both cell types were affected, or the ONL change was secondary to RPE loss or dysfunction. Ocular inflammation (uveitis) was reported to be frequent (75%) and at times severe after subretinal or intravitreal delivery of rAAV2.RPE65 in a study of RPE65mutant dogs [4]. Inadequately purified vector caused severe ocular inflammation in four dogs of our previous study [8]. In the present work, dogs also had clinical and histopathological signs of ocular inflammation, but this was relatively mild and slowly decreased over the postoperative period. Before use, our vector preparations were confirmed to be sterile and endotoxin free and, by silverstained PAGE analysis shown to contain only the three AAV capsid proteins [8]. There is evidence that intravitreal delivery of AAV2 leads to detection of transgene product in the visual pathways of the central nervous system [12,19,24–26]. Intravitreal AAV2-mediated gene therapy has been used in murine models of central neurodegenerative disease purposely to deliver the gene product to the brain [26,27]. Intravitreal AAV-2/2.gfp injection in three normal dogs was reported to result in detection of vector sequences in various regions of the visual pathways [12]. As has been affirmed [19], it is naRve to assume that subretinal delivery of a viral vector does not invariably have an intravitreal component, either at the time of surgery or due to postsurgical leakage back through the retinal perforation. Our inadvertent intravitreal or partly intravitreal injections of rAAV-2/2.RPE65 in RPE65mutant dogs may thus provide useful large animal data. Interestingly, in these cases there were no detectable vector sequences in postretinal visual centers in the brain. All but two subretinal injections also had no vector sequences in the postretinal visual pathways and in those that were positive, the levels of detectable vector were near the detection limit and could not be found upon repeat testing. Data from our rat biodistribution studies at both short and longer intervals suggested that vector spread outside the treated eye

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was limited and independent of whether the delivery was subretinal or intravitreal. What have we learned from the current study that can be translated to future human trials of RPE65associated LCA to minimize toxicity? First, we must carefully attend to details of the surgical technique. There should be positioning of the injection site so the expected retinal scarring from the needle tract does not compromise potentially functional retina. In vivo highresolution retinal microscopy in humans with RPE65associated LCA has indicated variation in the amount of photoreceptor layer structure remaining. Treatment sites should thus be individualized and based on pretreatment quantitation of retinal structure [11]. Second, based on the dog results, relative doses of rAAV-2/ 2.RPE65 between 0.1 and 0.3 delivered subretinally provided efficacy but no observed toxicity. Further safety studies are warranted to determine if the potential toxicity at the highest vector doses, based on retinal histopathological studies in the dog, is confirmed in nonhuman primates.

MATERIALS AND METHODS Animals and ocular surgery RPE65-mutant dogs (ages 2.7–7.4 months at injection; Table 1) and Sprague–Dawley rats (age 8 weeks at injection) were used. Ocular surgical procedures have been published [3,5,7,8,28]. All studies abided by the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research and institutional approval. rAAV-RPE65 Vector production, purification, and characterization Vector preparations were produced by the plasmid cotransfection method [29]. Briefly, one cell factory (Nalge Nunc International, Rochester, NY, USA) with approximately 1  109 HEK 293 cells is cultured in Dulbecco’s modified Eagle’s medium (cDMEM) supplemented with 5% fetal bovine serum and antibiotics. A CaPO4 transfection precipitation is set up by mixing a 1:1 molar ratio of rAAV vector plasmid DNA and serotype specific rep–cap helper plasmid DNA. This precipitate is added to 1100 ml of cDMEM and the mixture is applied to the cell monolayer. The transfection is allowed to incubate at 378C for 60 h. The cells are then harvested and lysed by three freeze/ thaw cycles. The crude lysate is clarified by centrifugation and the resulting vector-containing supernatant is divided among four discontinuous iodixanol step gradients. The gradients are centrifuged at 350,000g for 1 h, and 5 ml of the 60–40% step interface is removed from each gradient and combined. This iodixanol fraction is further purified and concentrated by column chromatography on a 5-ml HiTrap Q Sepharose column using a Pharmacia AKTA FPLC system (Amersham Biosciences, Piscataway, NJ, USA). The vector is eluted from the column using 215 mM NaCl, pH 8.0, and the rAAV peak collected. Vector-containing fractions are then concentrated and buffer exchanged in either Alcon BSS or AAV storage buffer (500 ml of Hyclone DPBS and 270 ml of 250 mM NaCl in H2O) using a Biomax 100K concentrator (Millipore, Billerica, MA, USA). Vector is then titered for DNase-resistant vector genomes by quantitative PCR relative to a standard. Finally, the purity of the vector is validated by silver-stained SDS–PAGE (the three AAV capsid proteins are the only visible protein bands in an acceptable prep), assayed for sterility and lack of endotoxin, and then aliquoted and stored at 808C. Clinical assessments Mortality and clinical observations. See supplementary material.

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Ophthalmic examinations. Dogs were examined prior to dosing, at 1 week postinjection, and then at 1, 2, and 3 months using slit-lamp biomicroscopy, indirect ophthalmoscopy, and measurement of intraocular pressure (Tono-Pen VET, Medtronics, Jacksonville, FL, USA). Electroretinography Dogs had full-field bilateral simultaneous ERGs using methods previously described [3,8,30,31]. Clinical pathology Peripheral venous blood was collected from dogs for hematology parameters and serum chemistries [32,33]. See supplementary material. Canine Anti-AAV2 antibody assays Plates (Dynex Immulon, 4HBX; Dynatech Laboratories, Inc., Chantilly, VA, USA) were coated with 1.29  109 AAV2 particles per well overnight at 48C in coating buffer (0.1 M NaHCO3, pH 8.4) and blocked at 378C for 2 h with 10% FBS (Cellgro; Mediatech, Inc., Herndon, VA, USA). Canine sera known to be positive against AAV2 were used as a standard and unknowns (the experimental dog sera) were serially diluted and incubated overnight at 48C. Detection antibody, HRP-conjugated rabbit anti-canine Ig (Sigma), was added to the wells at a 1:10,000 dilution and incubated at 378C for 2 h. Detection was accomplished with TMB peroxidase substrate (KPL) and the reaction stopped with 1 M H3PO4. The plate was read at 450 nm and data were tabulated relative to a standard curve using the seropositive reference. Necropsy and pathology See supplementary material. Canine ocular pathology Eye tissue was fixed in Bouin’s solution, embedded in paraffin, and cut for histological preparation by Research Pathology Services (Doylestown, PA, USA). Sections were taken along the vertical plane at four locations: through the optic nerve, at the nasal and temporal edge of the tapetal zone, and at a midpoint in the nasal retina between the optic nerve and the nasal edge of the tapetum. Hematoxylin- and eosin-stained 5-Am sections were examined for histopathology and select sections were used for immunocytochemistry using published methods [8]. Vertical retinal sections crossing the optic nerve were used for morphometry [34]. Contiguous fields, extending from the center of the optic nerve into the far superior and inferior retina, were imaged at 20 magnification using a video camera attached to a microscope. The resulting video was fed into a VCR (HR-S4800U, JVC, Japan) and a computer, and a video digitizer (PIXCI SV4 board, software version 2.1; EPIX, Inc., Buffalo Grove, IL, USA) was used to digitize selected images. Ten viewing field images extending from the center of the optic nerve into the superior and inferior retina were digitized and scaled using a calibration tool (Graticules, Ltd., Tonbridge, Kent, UK) imaged under the same magnification. A minimum of three ONL thickness measurements were made in locations within each field in which entire columns of nuclei could be identified. The average of measures for each location was used for analysis. Profiles of ONL thickness as a function of distance from the optic nerve were plotted for each eye. The locations of the subretinal injection and RPE65 expression (when available) in each profile were estimated using in vivo fundus drawings and immunocytochemical analyses from vertical histological sections, respectively. A control ONL thickness profile, created by averaging ONL thickness measurements from uninjected regions of control eyes, was used to estimate relative ONL thickness as a percentage of control at each sampled location in each eye. Relative ONL thickness values corresponding to injected and uninjected regions in each eye were compared using Student’s t test. Canine and rat biodistribution The spread of vector DNA in tissues was determined in samples collected at termination. For the dogs, tissues were collected with appropriate

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precautions to avoid any cross-contamination, snap frozen in liquid nitrogen, and transferred immediately to 808C storage where they remained until the time of DNA extraction using the Qiagen DNeasy (Qiagen, Inc., Valencia, CA, USA) tissue extraction kit. Total copies of the rAAV genome in dog tissue samples were quantified by real-time PCR using an ABI 7700 Sequence Detection System (Applied Biosystems, Foster City, CA, USA) and analyzed using the SDS 2.0 software. Varying amounts of DNA were used in the PCRs depending on the total amounts available from each tissue. A set of spiked reactions was performed using tissues from one male and one female animal prior to assaying the remaining tissues. For the rat studies, tissues (see supplementary tables) were collected for biodistribution along with a portion of the injected eye with attached intraorbital optic nerve. Precautions were taken to avoid cross-contamination while harvesting tissues. Once removed, each tissue was held over a petri dish and rinsed with sterile 1 PBS. The tissue was placed on a weigh boat and weighed (if applicable). Samples were then placed into 2-ml screw-cap tubes and immersed in liquid nitrogen where they remained until analysis and then were transferred to storage at 808C. Genomic DNA (gDNA) was extracted from tissues according to the manufacturer’s protocol (Qiagen DNeasy tissue kit). Briefly, up to 20 mg tissue (100 Al blood) was digested overnight in proteinase K. Resulting DNA concentrations from the extraction procedure were determined using an Eppendorf Biophotometer (Model 6131; Eppendorf, Hamburg, Germany). One microgram of extracted gDNA was used in all quantitative PCRs according to a previously used protocol [35,36] and reaction conditions follow those recommended by Perkin–Elmer/Applied Biosystems and include 50 cycles of 948C for 40 s, 378C for 2 min, 558C for 4 min, and 688C for 30 s. Primer pairs were designed to the CMV enhancer/chicken h-actin promoter as described [37] and standard curves established by spike-in concentrations of a plasmid DNA (CBAT) containing the same promoter as above and the a1-antitrypsin cDNA [35]. DNA samples were assayed in triplicate. The third replicate was spiked with CBAT DNA at a ratio of 100 copies/Ag of gDNA. If at least 40 copies of the spike-in DNA were detected, the DNA sample was considered acceptable for reporting vector DNA copies. When the copy number of the vector DNA found in that sample was greater than 100 copies/Ag the sample was considered positive and the measured copy number/Ag reported. If fewer than 100 copies/Ag were present, the sample was considered negative. When less than 1 Ag of gDNA was analyzed to avoid PCR inhibitors copurifying with DNA in the extracted tissue, the spike-in copy number was reduced proportionally to maintain the 100 copies/Ag DNA ratio.

ACKNOWLEDGMENTS We thank Amanda Nickle and the staff of the RDS facility, Elaine Smilko, Andy Cheung, Michelle Doobrajh, Paul Schied, Heather DeHeer, Jane Bauman, Amy Poirier, Lynn Combee, Kirsten Erger, Cheryl Roberts, Arun Mani, Cathy Hoover, and Margaret Humphries. The work was supported by the NIH/NEI (EY-13729, EY-13385, EY-06855, EY-11123, EY-13132, EY-08061), Macula Vision Research Foundation, Foundation Fighting Blindness, Macular Disease Foundation, Mackall Trust, ONCE International Prize for R&D in Biomedicine and New Technologies for the Blind, and the F. M. Kirby Foundation. RECEIVED FOR PUBLICATION OCTOBER 24, 2005; REVISED FEBRUARY 28 2006; ACCEPTED MARCH 1, 2006.

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