Long-term Retinal Function and Structure Rescue Using Capsid Mutant AAV8 Vector in the rd10 Mouse, a Model of Recessive Retinitis Pigmentosa

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Long-term Retinal Function and Structure Rescue Using Capsid Mutant AAV8 Vector in the rd10 Mouse, a Model of Recessive Retinitis Pigmentosa
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  srcinal article  234 www.moleculartherapy.org vol. 19 no. 2, 234–242 feb. 2011 © The American Society of Gene & Cell Therapy The retinal degeneration 10 ( rd10 ) mouse is a well-charac-terized model of autosomal recessive retinitis pigmentosa (RP), which carries a spontaneous mutation in the β  subunit of rod cGMP-phosphodiesterase (PDE β ). Rd10  mouse exhibits photoreceptor dysfunction and rapid rod photoreceptor degeneration followed by cone degen-eration and remodeling of the inner retina. Here, we evaluate whether gene replacement using the fast-acting tyrosine-capsid mutant AAV8 (Y733F) can provide long-term therapy in this model. AAV8 (Y733F)-smCBA-PDE β  was subretinally delivered to postnatal day 14 (P14) rd10  mice in one eye only. Six months after injection, spec-tral domain optical coherence tomography (SD-OCT), electroretinogram (ERG), optomotor behavior tests, and immunohistochemistry showed that AAV8 (Y733F)- mediated PDE β  expression restored retinal function and visual behavior and preserved retinal structure in treated rd10  eyes for at least 6 months. This is the first demonstra-tion of long-term phenotypic rescue by gene therapy in an animal model of PDE β -RP. It is also the first example of tyrosine-capsid mutant AAV8 (Y733F)- mediated correc-tion of a retinal phenotype. These results lay the ground-work for the development of PDE β -RP gene therapy trial and suggest that tyrosine-capsid mutant AAV vectors may be effective for treating other rapidly degenerating models of retinal degeneration. Received 23 August 2010; accepted 10 November 2010; published online 7 December 2010. doi:10.1038/mt.2010.273 INTRODUCTION Retinitis pigmentosa (RP) is a family of inherited diseases caus-ing retinal degeneration and eventual photoreceptor cell death. Visual loss in RP patients is progressive, with rod photoreceptor degeneration typically preceding cone photoreceptor loss. 󰀀ere is currently no treatment for this debilitating disorder. A common form of autosomal recessive RP has been linked to mutations in the β-subunit of rod cGMP-phosphodiesterase (PDEβ), which is encoded by the Pde6b  gene. 1  cGMP-PDE controls the levels of cGMP and Ca 2+  in rod outer segments. Improper function of cGMP-PDE causes an accumulation of cGMP within photo-receptors and prevents these cells from processing input light effi-ciently. Accounting for ~5% of all cases, Pde6b -RP is one of the earliest onset and most aggressive forms of this disease. 1–3 󰀀erapeutic approaches have been tested in two mouse models of RP carrying different mutations in Pde6b , the rd1  and retinal degeneration 10 ( rd10)  mice. Progress in developing a therapy has been hampered by the very rapid retinal degeneration seen in the rd1  model that initiates at or before photoreceptor outer seg-ments even form. 4–6  Natural history studies suggest that the rd10  mouse, which carries a point mutation in exon 13 of Pde6b , better emulates the progression of typical human autosomal recessive RP than the previously described rd1  mouse. 7–9  Loss of photorecep-tors in the rd10  mouse begins around P18, with peak photorecep-tor death occurring at P25. 9  Most photoreceptor cells are lost by 5 weeks. 7–9  Similar to that seen in the rd1  mouse, 10,11  this cell death in rd10  is accompanied and followed by structural and functional changes in the inner retina. 9  Such remodeling has also been docu-mented in RP patients. 12  Remodeling occurs primarily in bipolar and horizontal cells by progressive dendritic retraction and loss of synaptic connections as photoreceptors initiate cell death, and is followed by glial and amacrine cell remodeling 9–11,13–21  with ganglion cell sparing. 22–24  In addition, some neuronal processes become hypertrophic and sprout ectopically. Ultimately, bipolar and horizontal cells are also lost. Interestingly, when photorecep-tor death coincides with outer retinal synaptogenesis, dendrites do not develop properly, suggesting a possible trophic depen-dence of second order neurons on the presence of photorecep-tors for maintenance of their proper morphology. 10  󰀀us, gene therapy-mediated preservation of photoreceptors, even relatively The first two authors contributed equally for this work. Correspondence:  Ji-jing Pang, Eye Hospital, School of Ophthalmology & Optometry, Wenzhou Medical College, Wenzhou, China 325027. E-mail:  jpang@ufl.edu  Long-term Retinal Function and Structure Rescue Using Capsid Mutant AAV8 Vector in the rd10   Mouse, a Model of Recessive Retinitis Pigmentosa Ji-jing Pang 1,2 , Xufeng Dai 1,2 , Shannon E Boye 2 , Ilaria Barone 3 , Sanford L Boye 2 , Song Mao 2 , Drew Everhart 4 , Astra Dinculescu 2 , Li Liu 2 , Yumiko Umino 4 , Bo Lei 5 , Bo Chang 6 , Robert Barlow 4,* , Enrica Strettoi 3  and William W Hauswirth 2 1 Eye Hospital, School of Ophthalmology & Optometry, Wenzhou Medical College, Wenzhou, Zhejiang, China; 2 Department of Ophthalmology, University of Florida, Gainesville, Florida, USA; 3 CNR Neuroscience Institute, Pisa, Tuscany, Italy; 4 The Center for Vision Research, State University of New York Upstate Medical University, Syracuse, New York, USA; 5 Ophthalmology, the First Affiliated Hospital of Chongqing Medical University, Chongqing Key Laboratory of Ophthalmology, Chongqing, China; 6 The Jackson Laboratory, Bar Harbor, Maine, USA. *Deceased    Molecular erapy vol. 19 no. 2 feb. 2011 235 © The American Society of Gene & Cell Therapy Mutant AAV8-mediated Long-term Rescue in rd10  Mice late in the course of degeneration may have a positive effect on inner retinal remodeling. Notably, photoreceptor cell loss occurs aer terminal differentiation of the rd10  retina. 󰀀us, the rate of photoreceptor loss in the rd10  mouse is substantially delayed and slower than that seen in the rd1  retina. Rearing rd10  mice in dark-ness further slows the rate of retinal degeneration by as much as 4 weeks. 8,25  Taken together, these studies suggest the rd10  mouse is a better model in which to test RP therapeutic strategies. Various approaches for preserving retinal structure and function in rd10  mice have been used including stem cell, antiapoptotic, antioxi-dant and gene-replacement therapies, 25–27  all of which have shown only a partial and/or short-term measure of retinal rescue.We have previously shown that serotype 5, adeno-associated  virus (AAV) vector containing a minimal chicken β actin pro-moter/CMV enhancer (smCBA) was capable of delivering func-tional PDEβ to the rd10  retina. 25  Following P14 treatment with this photoreceptor-preferential AAV serotype, we showed that rod and cone-mediated function and structure were preserved for at least 3 weeks. 25  However, by 6 weeks post-treatment, therapeutic effects had faded. Because AAV5-mediated transgene expression occurs 1–2 weeks aer injection in the retina 25  and photorecep-tor apoptosis initiates around P16–P18 in the rd10  mouse, it is possible that P14 treatment with standard AAV5 did not elicit sufficient PDEβ expression early enough in the rd10  retina to arrest the degenerative progression, and therefore was insufficient to provide long-lasting therapeutic benefit.Recent studies showed that the photoreceptor-preferential serotype 8 AAV (AAV8) vector was effective at restoring retinal structure and function to animal models of retinal degeneration which were previously shown to be refractory to AAV2 or AAV5 treatment. 28,29  Investigators attributed this to the more rapid onset of transgene expression mediated by AAV8 relative to other serotypes. 30  In addition, recent reports demonstrate that phos-phorylation of tyrosine residues in AAV capsid proteins greatly affect the transduction characteristics of this vector system. 31,32  It has been shown that inhibiting phosphorylation results in strengthening of transgene expression levels and an increase in transduction kinetics relative to standard AAV. 33  󰀀e proposed mechanism is that reduced phosphorylation of surface tyrosine residues decreases ubiquitination and proteosome-mediated deg-radation, thereby facilitating nuclear transport of AAV vector. 31  Subsequent to this finding, AAV2 vectors with surface tyrosine residues mutated to phenylalanine (Y to F) were shown to have increased transduction characteristics relative to unmodified capsid in vitro  and in murine hepatocytes. 32  Bearing these results in mind, we have previously shown that serotype 8 AAV contain-ing a single point mutation (Y733F) in a surface-exposed tyrosine residue confers earlier onset and stronger transgene expression in photoreceptor cells than standard AAV8. 33  Here we report that strong and fast-acting AAV8-733 vector mediating PDEβ expres-sion is capable of preserving retinal structure and function in the rd10  retina for the long term. 󰀀is is the first demonstration of long-term restoration of vision by gene therapy in an animal model of Pde6b -RP. In addition, this is the first demonstration that an AAV8 tyrosine-capsid mutant is capable of conferring more effective therapy than that of a standard AAV vector in an animal model of retinal disease. 󰀀ese results lay the groundwork for the development of an AAV-based gene therapy vector for treatment of patients with the Pde6b  form of RP and support the use of AAV tyrosine-capsid mutants for the treatment of other rapidly degen-erating models of retinal degeneration. RESULTS Optomotor behavior reveals the efficacy of AAV8 (Y733F)-smCBA-PDE β  for rescue of both photopic and scotopic vision in treated rd10   eyes Optomotor behavioral analysis showed that uninjected wild-type eyes ( Figure 1 , black bars, n  = 4) responded significantly better than untreated rd10  eyes (black bars, n  = 4, P   < 0.05), but similar to treated rd10  eyes (gray bars, n  = 4, P   > 0.05) under all con-ditions. Under dark-adapted conditions ( Figure 1 , top), treated rd10  eyes had an acuity of 0.493 ± 0.089 cycles/degree (cyc/deg) (white bar, n  = 4), a level essentially identical to that of wild-type eyes (0.499 ±  0.039 cyc/deg, black bar, n  = 4) and significantly better than that of untreated rd10  eyes (gray bar, 0.098 ±  0.087, n  = 4, P   < 0.001). Dark-adapted contrast sensitivities ( Figure 1 ) were similar to scotopic acuity results; treated rd10  eyes (contrast sensitivity of 6.338 ±  4.246, n  = 4) showed contrast thresholds similar to uninjected wild-type eyes (4.398 ±  0.739, n  = 4, P   = 0.425), but performed significantly better than untreated rd10  eyes, which displayed a contrast sensitivity of 1.05 ±  0.03 ( n  = 4, P   < 0.05). In all scotopic tests, untreated rd10  eyes performed extremely poorly, essentially equivalent to a lack of rod-mediated  visual function. Under photopic, cone-dominated conditions, wild-type eyes showed an average acuity of 0.499 ±  0.39 cyc/deg ( Figure 1 , black bar, n  = 4) and an average contrast sensitivity of 8.793 ±  2.790 ( Figure 1 , black bar, n  = 4). Treated rd10  eyes exhib-ited improved acuity (0.493 ±  0.089 cyc/deg, n  = 4, P   < 0.001) and contrast sensitivity (20.405 ±  7.196, n  = 4, P   < 0.001) relative to untreated rd10  eyes (acuity = 0.098 ±  0.087 cyc/deg and contrast sensitivity= 1.075 ±  0.053, n  = 4 each). Statistical comparisons of all behavioral measurements are shown in Table 1 . AAV tyrosine-capsid mutant vector AAV8 (Y733F) restores retinal function (ERG) to the rd10   mouse in the long term whereas standard AAV5 and AAV8 vectors do not Six months following treatment with AAV8 (Y733F)-smCBA-PDEβ, rd10  mice were examined by dark- and light-adapted elec-troretinograms (ERGs). Most of the dark-adapted ( Figure 2a ) and the majority of the light-adapted ( Figure 2b ) ERG signal amplitudes were maintained in treated rd10  eyes. In contrast, ERG signals under both conditions were almost undetectable in untreated rd10  eyes. Rescue was most evident in eyes that experienced the largest reti-nal detachment during vector administration and the least surgical complications (data not shown). Restored scotopic ERGs exhibited typical a-wave structure and the average b-wave amplitudes at 0.4 log cd-s/m 2  intensity were 501.67 ±  21.19 ( n  = 3), 289.87 ±  26.24 ( n  = 3), and 12.57 ±  4.20 ( n  = 3) in normal uninjected C57BL/6J, treated and untreated rd10  eyes, respectively ( Figure 2c ). Photopic ERG amplitudes at 1.4 log cd-s/m 2  intensity were 106.67 ±  7.12 ( n  = 3), 81.37 ±  7.12 ( n  = 3) and 8.13 ±  4.74 ( n  = 3) in normal C57BL/6J, treated and untreated rd10  eyes, respectively ( Figure 2d ). Dark-and light-adapted b-wave amplitudes in treated rd10  eyes were ~58%  236 www.moleculartherapy.org    vol. 19 no. 2 feb. 2011 © The American Society of Gene & Cell Therapy Mutant AAV8-mediated Long-term Rescue in rd10  Mice and 68% of those from age-matched, uninjected C57BL/6J eyes. A paired t  -test analysis revealed a significant difference between b-wave amplitudes of AAV8(Y733F)-smCBA-PDEβ-treated and untreated rd10  eyes under both dark- and light-adapted conditions ( P   < 0.01) although a statistical difference was also found between normal uninjected C57BL/6J and treated rd10  eyes ( P   < 0.05). To test the sensitivity of the rod and cone system in treated eyes, we measured the implicit time of the rod- and cone-driven b-waves. We found no significant difference in implicit times of the rod-driven b-waves between the treated rd10  eyes (74.33 ±  4.04 ms) and C57BL/6J eyes (72.33 ±  2.08 ms, n  = 3, P   = 0.597). Likewise, no sig-nificant difference was found in implicit times of the cone-driven b-waves between the treated rd10  (56.67 ±  5.77 ms) and C57BL/6J eyes (53.33 ±  5.77 ms, n  = 3, P   = 0.423).To highlight the effectiveness of AAV8 (Y733F)-mediated treat-ment relative to standard photoreceptor-selective serotypes, AAV5 and AAV8, we compared rod and cone-mediated ERG amplitudes in mice injected at P14 with different vectors at 4 weeks, 8 weeks, and 6 months aer treatment ( Figure 3 ). At 4 weeks aer treat-ment, rod and cone-mediated ERG responses were highest in rd10  mice treated with AAV8 (Y733F)-smCBA-PDEβ ( Figure 3 , le). 󰀀is mutant serotype elicited even higher response amplitudes over the next 4 weeks as indicated in the 8 weeks aer measure-ments ( Figure 3 , middle). At 8 weeks aer treatment, rod and 0.500.250.00    A  c  u   i   t  y   A  c  u   i   t  y Dark adapted acuityLight adapted acuityC57 rd10T UC57 rd10T UC57 rd10T UC57 rd10T U0.250.500.00Dark adapted contrastsensitivityLight adapted contrastsensitivity101110    C  o  n   t  r  a  s   t  s  e  n  s   i   t   i  v   i   t  y   C  o  n   t  r  a  s   t  s  e  n  s   i   t   i  v   i   t  y Figure 1 AAV8 (Y733F)-smCBA-PDE β  treatment at P14 restores both scotopic and photopic visual acuity and contrast sensitivity in rd10   eyes.  Under dark-adapted (top) or light-adapted (bottom) conditions, the visual acuity (left) and contrast sensitivity (right) in treated (T) rd10  eyes (T, n  = 4) are similar to those of wild-type animals (C57). Untreated (U) rd10  eyes (U, n  = 4) show very poor acuity and contrast sensitivity under either dark- or light-adapted conditions. 3002001000 − 1001000 − 100Scotopic ERG0 50 100 150 2000 50 100 150 2007550250 − 25250 − 25 − 50 Photopic ERG6005004003002001000120100806040200Normal Treated UntreatedNormal Treated Untreated abcd Figure 2 Scotopic and photopic electroretinogram (ERG) in treated and untreated rd10   eyes. ( a ) Representative scotopic ERG elicited from 0.4 log cd-s/m 2  intensity in an rd10  eye 6 months following treatment at P14 (upper) compared with that of the untreated eye (lower) from the same rd10  mouse; y axis: 20 µV/Div, x axis: 50 ms/Div. ( b ) Representative photopic ERGs elicited at 1.4 log cd-s/m 2  from the same rd10  mouse (upper, treated; lower, untreated); y axis: 5 µV/Div, x axis: 10 ms/Div. ( c ) Average scotopic b-wave amplitudes elicited at 0.4 log cd-s/m 2  in age-matched, uninjected normal C57 BL/6J (left), treated (middle), and untreated (right) rd10  eyes; ( d ) Averaged photopic b-wave amplitudes elicited at 1.4 log cd-s/m 2  in age-matched, uninjected normal C57 BL/6J (left), treated (middle), and untreated (right) rd10  eyes. Table 1 Statistical comparison of the scotopic and photopic visual function of wild type, P14 vector-treated and untreated rd10   mouse eyes as measured by optomotor behaviorDark-adapted acuity ( n  = 4)Dark-adapted contrast sensitivity ( n  = 4)C57BL/6JTreated rd10Untreated rd10C57BL/6JTreated rd10Untreated rd10 0.355 ±  0.0390.377 ±  0.0420.069 ±  0.0284.398 ±  0.7396.338 ±  4.2461.028 ±  0.025C57BL/6J versus treated rd10: P   = 0.5306Treated versus untreated rd10: P   = 0.0002***C57BL/6J versus treated rd10: P   = 0.425Treated versus untreated rd10: P   = 0.044* Light-adapted acuity ( n  = 4)Light-adapted contrast sensitivity ( n  = 4)C57BL/6JTreated rd10Untreated rd10C57BL/6JTreated rd10Untreated rd10 0.499 ±  0.0390.493 ±  0.0890.098 ±  0.0878.793 ±  2.79020.405 ±  7.1961.075 ±  0.053C57BL/6J versus treated rd10: P   = 0.9230Treated versus untreated rd10: P   = 0.0008 ***C57BL/6J versus treated rd10: P   = 0.097Treated versus untreated rd10: P   = 0.0006*** Under scotopic and photopic conditions, AAV8 (Y733F)-smCBA-PDE β  treatment of rd10  eyes produces visual acuity and contrast sensitivity similar to wild-type animals. Each mouse was tested for four to six trials per condition. Data from each cohort of animals ( n  = 4) were then averaged to obtain the means for each test condition. The standard deviation listed is the standard deviation of the individual mouse means (mean ±  SD). P   values were calculated using a paired two-tailed t  -test. *** P   < 0.001, * P   < 0.05.   Molecular erapy vol. 19 no. 2 feb. 2011 237 © The American Society of Gene & Cell Therapy Mutant AAV8-mediated Long-term Rescue in rd10  Mice  cone-mediated function was undetectable in AAV5-treated rd10  mice, whereas AAV8-treated rd10  mice still displayed measur-able rod-mediated function and robust cone-mediated function ( Figure 3 , middle). By 6 months aer treatment, rod and cone responses were undetectable in AAV8-treated rd10  mice ( Figure 3 , right). AAV8 (Y733F) was the only vector which elicited rod and cone-mediated ERG responses at 6 months aer treatment indi-cating that this serotype is superior to standard AAV5 and AAV8  vectors in its ability to restore long-term and stable function to pho-toreceptors in the rd10  mouse. Similar results were also obtained from those rd10  mice raised in normal 12 hours/12 hours vivarium cycling room light ( Supplementary Figure S1 ). AAV8 (Y733F)-smCBA-PDE β  preserves retinal structure in treated rd10   eyes Spectral domain optical coherence tomography (SD-OCT) allows retinal thickness assessment in treated versus untreated eyes, in vivo . OCT examination revealed that AAV8 (Y733F)-mediated PDEβ expression is capable of preserving retinal morphology in treated rd10  retinas. In order to compare the retinal thickness of treated and untreated retinas of the same mouse in an unbiased manner, a representative rd10  mouse that displayed >90% retinal detachment in its treated eye (considered a very good subreti-nal injection) was carefully measured in eight comparable reti-nal areas, 3 mm from the optic nerve (ON) head in both treated and untreated eyes. Comparisons of retinal thickness revealed that the thickness of untreated rd10  retina was 0.108 ±  0.007 mm ( n  = 8), while it was 0.138 ±  0.011 mm ( n  = 8, P   < 0.001) in treated rd10  retina. 󰀀is difference in retinal thickness is clearly visible in a representative image taken 3 mm temporal of the ON head ( Figure 4a ).Studies have shown that OCT measurements of retinal thick-ness correspond to follow-up histological measurements. 34  Indeed, following killing and enucleation at 6 months aer treatment, 4003503002502001501005004003503002502001501005004003503002502001501005001201008060402001 2 3 1 2 3 1 2 31 2 31201008060402001 2 31201008060402001 2 3AAV51: AAV82: AAV8 (Y733F)3: P14+ 4 weeks P14+ 8 weeks P14+ 6 months    S  c  o   t  o  p   i  c   b  -  w  a  v  e  a  m  p   l   i   t  u   d  e  s   (      µ    V   )   P   h  o   t  o  p   i  c   b  -  w  a  v  e  a  m  p   l   i   t  u   d  e  s   (      µ    V   ) Figure 3 Comparisons of retinal function in adeno-associated virus vectors of serotype 5 (AAV5), AAV8, and AAV8 (Y733F)-treated rd10   mice over time.  (1) AAV5, (2) AAV8, (3) AAV8 (Y733F)-mediated gene therapy all have rescue effects 4 weeks after P14 treatment both in sco-topic (upper, left) and photopic (lower, left) conditions. AAV8 and AAV8 (Y733F) both have rescue effects 8 weeks following injections (middle column). Only AAV8 (Y733F) has rescue effect 6 months following P14 treatment both in scotopic (upper, right) and photopic (lower, right) conditions. Treated rd10 eye Untreated rd10 eye abc 500   µ m Figure 4 Bioptigen spectral domain optical coherence tomography (SD-OCT) and light microscopic (LM) images of treated (left) and untreated (right) eyes from one rd10   mouse.  ( a ) OCT images at same location (3 mm temporal to the optic nerve) from representative treated and untreated rd10  eyes. Scale calipers on each image are placed at equivalent distances from the optic nerve to quantify the distance from the vitreal face of the ganglion cell layer to the apical face of the RPE (152 millimeters for the treated eye and 102 millimeters for the untreated eye). ( b ) Low-magnification images from the treated and untreated eyes of the same rd10  mouse used in a . Note the presence of outer segments and continuous, intact outer nuclear layer (ONL) in the treated eye and their absence in the untreated eye. ( c ) High magnification LM images  from the treated and untreated eyes of the same rd 10  mouse used in b . OS, outer segments; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner pexiform layer; RGC, retinal ganglion cell.  238 www.moleculartherapy.org    vol. 19 no. 2 feb. 2011 © The American Society of Gene & Cell Therapy Mutant AAV8-mediated Long-term Rescue in rd10  Mice hematoxylin and eosin staining of AAV8 (Y733F)-smCBA-PDEβ-treated rd10  retinas and contralateral, untreated controls con-firmed our OCT results. Retinal structure was largely maintained in treated rd10  eyes; the outer nuclear layer (ONL) was about 5–8 nuclei thick compared to the 7–10 layers in uninjected C57BL/6J eyes (data not shown). Only one layer of nuclei remained in the ONL of untreated rd10  eyes. Using a retina from a representative rd10  mouse which received >90% retinal detachment postinjec-tion, light microscopy at low-magnification revealed a relatively normal ONL throughout the retina ( Figure 4b , le). In contrast, it was difficult to visualize the ONL in untreated eye of the same rd10  mouse ( Figure 4b , right). Higher-magnification images showed that up to 50% of the normal outer segment length and 70% of normal ONL thickness were maintained in the treated rd10  retina ( Figure 4c , le). Meanwhile, there was only one incomplete layer of nuclei in the ONL of the untreated eye from the same rd10  mouse ( Figure 3c , right). In addition, the outer plexiform layer and inner nuclear layer were also clearly thinned in untreated rd10  retina ( Figure 4c , right) relative to that seen in the partner treated eye ( Figure 4c , le). Abundant AAV8 (Y733F)-mediated PDE β  expression detected in retinas of treated rd10   mice Six months aer treatment, PDEβ expression was assayed by immu-nohistochemistry of treated and untreated retinal sections from rd10  mice. As seen in normal C57BL/6J retina ( Figure 5a ), strong PDEβ staining is evident in the outer segments of a representative treated rd10  retina ( Figure 5a ) while no PDEβ expression was observed in the partner untreated retina ( Figure 5c ) from the same rd10  mouse. Western blot analysis confirmed the presence of the ~90 kDa PDEβ protein in the treated rd10  retina only ( Figure 5d ). In addition, expression of another phototransduction protein, rod transducin α was also measured by immunoblot and served as another biochemical indicator of photoreceptor cell survival. Our results show that rod transducin α was maintained in treated rd10  eyes, but not in untreated, contralateral controls in which photore-ceptor degeneration is virtually complete ( Figure 5e ). AAV8 (Y733F)-mediated PDE β  expression prevents secondary neuronal remodeling in the rd10   retina Immunocytochemistry confirmed retention of bipolar, horizon-tal, amacrine, and cone cell morphology in treated rd10  retinas at 6 months aer treatment ( Figure 6 ). Cone opsin staining revealed maintenance of well organized cone outer segments in treated eyes whereas its expression was restricted to the cell bodies of aberrant cones in untreated controls ( Figure 6 , le column). As in wild-type controls, photoreceptor synapses in treated rd10  retinas were abundant ( Figure 6 , middle column). In con-trast to untreated eyes, dendrites of rod bipolar cells were well-elaborated and penetrated the cavity of rod spherules in treated retinas ( Figure 7 , right column). Horizontal cell processes were observed in close proximity to the synaptic terminal of rods and cones ( Figure 7 , le column). In contralateral, untreated retinas, second order neurons remodeled upon photoreceptor degenera-tion, undergoing dendritic atrophy ( Figures 6  and 7 ). Lamination of AAV8 (Y733F)-treated rd10  retinas was preserved and all syn-aptic markers produced a normal pattern of staining. DISCUSSION 󰀀is study is the first demonstration that long-term restoration of vision and preservation of retinal structure can be achieved in the rd10  mouse with gene therapy. In addition, it is the first example of AAV8 tyrosine-capsid mutant-mediated therapy in an animal model of disease. Notably, AAV8 (Y733F)-mediated PDEβ expression conferred more effective, long-term therapy to the rd10  mouse than that seen with standard AAV8 and AAV5 vectors. 25  Previous studies have shown that animal models exhibiting rapid retinal disease onset are refractory to AAV5-, but responsive to AAV8-mediated treatment, both photoreceptor-preferential AAV serotypes. 28  Investigators hypothesized that the onset of transgene expression mediated by AAV5, which is known to be slower rela-tive to AAV8, 30  was not sufficient to provide therapy in an animal model which exhibits very early pathology. Here we show that standard AAV8 was also unable to provide long-term therapy to the rd10  mouse. Arresting disease pathology in this RP mouse model requires very rapid onset of therapeutic PDEβ expression that is achievable only with the fast-acting AAV8 (Y733F) capsid mutant. Our ERG results ( Figure 3 ) highlight the relative abili-ties of each P14-delivered serotype to restore retinal function to the rd10  mouse. At 8 weeks aer treatment, retinal function was lost in AAV5-treated mice whereas both rod and cone- mediated function were still easily detectable in AAV8-treated mice. However, AAV8-treated rod responses at this time point were already decreasing relative to that seen at 4 weeks aer treatment, Treated UntreatedMW Treated UntreatedMW100 kd75 kdPDE6b50 kd37 kdTransducin de Figure 5 PDE β  expression in treated and untreated eyes of rd10   mice.  PDE β  immunostaining (red) in a 6.5-month old ( a ) uninjected normal C57BL/6J eye, ( b ) treated, and ( c ) untreated eye from one rd10  mouse. Nuclei were stained with DAPI (blue). Note the robust staining of PDE β  in outer segments of the treated rd10  eye compared to its absence in untreated, contralateral control eye. Western blot showing ( d ) abun-dant PDE β  and ( e ) rod transducin- α  expression in treated but not in untreated rd10  eyes. PDE β , β  subunit of rod cGMP-phosphodiesterase; OS, outer segments; IS, inner segments; ONL, outer nuclear layer.
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