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1.  Self-complementary AAV5 Vector Facilitates Quicker Transgene Expression in Photoreceptor and Retinal Pigment Epithelial Cells of Normal Mouse 
Experimental eye research  2010;90(5):546-554.
To clarify whether transduction efficiency and cell type specificity of self-complementary (sc) AAV5 vectors are similar to those of standard, single stranded AAV5 vectors in normal retina, one micro liter of scAAV5-smCBA-GFP vector (1X1012 genome containing particles/ml) and AAV5-smCBA-GFP vector (1X1012 genome containing particles/ml) were subretinally or intravitreally (in both cases through the cornea) injected into the right and left eyes of adult C57BL/6J mice, respectively. On post-injection day (PID) 1, 2, 5, 7, 10, 14, 21, 28 and 35, eyes were enucleated; retinal pigment epithelium (RPE) wholemounts, neuroretinal wholemounts and eyecup sections were prepared to evaluate green fluorescent protein (GFP) expression by fluorescent microscopy. GFP expression following trans-cornea subretinal injection of scAAV5-smCBA-GFP vector was first detected in RPE wholemounts around PID 1 and in neuroretinal wholemounts between PID 2 and 5; GFP expression peaked and stabilized between PID 10-14 in RPE wholemounts and between P14 and P21 in neuroretinal wholemounts with strong, homogeneous green fluorescence covering the entire wholemounts. The frozen sections supported the following findings from the wholemounts: GFP expression appeared first in RPE around PID 1-2 and soon spread to photoreceptors (PR) cells; by PID 7, moderate GFP expression was found mainly in PR and RPE layers; between PID 14 and 21, strong and homogenous GFP expression was observed in RPE and PR cells. GFP expression following subretinal injection of AAV5-smCBA-GFP was first detected in RPE wholemounts around PID 5-7 and in neuroretinal wholemounts around PID 7-10; ssAAV5 mediated GFP expression peaked at PID 21 in RPE wholemounts and around PID 28 in neuroretinal wholemounts; sections from AAV5 treated eyes also supported findings obtained from wholemounts: GFP expression was first detected in RPE and then spread to the PR cells. Peak GFP expression in RPE mediated by scAAV5 was similar to that mediated by AAV5. However, peak GFP expression mediated by scAAV5 in PR cells was stronger than that mediated by AAV5. No GFP fluorescence was detected in any retinal cells (RPE wholemounts, neuroretinal wholemounts and retinal sections) after trans-cornea intravitreal delivery of either scAAV5-GFP or AAV5-GFP. Neither scAAV5 nor AAV5 can transduce retinal cells following trans-cornea intravitreal injection. The scAAV5 vector used in this study directs an earlier onset of transgene expression than the matched AAV5 vector, and has stronger transgene expression in PR cells following subretinal injection. Our data confirm the previous reports that scAAV vectors have an earlier onset than the standard, single strand AAV vectors (Natkunarajah et al., 2008; Yokoi et al., 2007). scAAV5 vectors may be more useful than standard, single stranded AAV vector when addressing certain RPE and/or PR cell-related models of retinal dystrophy, particularly for mouse models of human retinitis pigmentosa that require rapid and robust transgene expression to prevent early degeneration in PR cells.
doi:10.1016/j.exer.2010.01.011
PMCID: PMC2854182  PMID: 20138034
transgene expression; self-complementary AAV; scAAV5; AAV5; subretinal injection; intravitreal injection; photoreceptor; RPE
2.  Intraocular route of AAV2 vector administration defines humoral immune response and therapeutic potential 
Molecular Vision  2008;14:1760-1769.
Purpose
Safety and efficiency are critical for successful gene therapy. Adeno-associated viral (AAV) vectors are commonly used for gene transfer in both human and animal studies. However, administration of AAV vectors can lead to development of neutralizing antibodies against the vector capsid, thus decreasing the efficiency of therapeutic gene transfer and preventing effective vector readministration. We investigated immune responses to different routes of ocular administration and readministration of AAV vectors, and the effect of previous exposure of AAV vector in one eye on the transduction efficacy of subsequent intraocular AAV-mediated gene delivery to the partner eye.
Methods
We tested two vector systems. One contained a cDNA encoding a secreted pigment epithelial derived factor (PEDF) cDNA under the control of a Cytomegalovirus (CMV) enhancer and chicken β-actin promoter (CBA; AAV2-CBA-PEDF) and was tested in a murine model of laser-induced choroidal neovascularization (CNV). The other vector contained a cDNA encoding the intracellular reporter green fluorescent protein (GFP) under the control of the same promoter (AAV2-CBA-GFP). Animals were divided into groups and received sequential injections at different combinations of either intravitreal or subretinal routes. CNV was evaluated by fluorescein angiographic choroidal flat-mount image analysis. The expression of GFP was analyzed in retinal sections by direct fluorescence imaging. Antibodies against AAV2 capsid and transgenes were analyzed by ELISA using serum samples collected before injection and different time points after the injection. Neutralizing antibodies were characterized by in vitro assays.
Results
Various ocular compartments responded to AAV administration differently. Intravitreal administration of AAV vectors, which resulted in transduction of inner retina (primarily retinal ganglion cells), generated a humoral immune response against AAV capsid that blocked vector expression upon readministration via the same route into the partner eye. In contrast, it had no effect on vector readministered into the subretinal space of the partner eye. Additionally, subretinal administration of vector did not trigger any humoral immune response against AAV capsid, and had no effect on subsequent administration of vector either intravitreally or subretinally into the partner eye.
Conclusions
These findings have important clinical implications for the design of AAV-mediated ocular gene transfer for retinal diseases, particularly if both eyes require sequential treatment.
PMCID: PMC2559816  PMID: 18836574
3.  Tyrosine capsid-mutant AAV vectors for gene delivery to the canine retina from a subretinal or intravitreal approach 
Gene therapy  2013;21(1):10.1038/gt.2013.64.
Recombinant adeno-associated viruses are important vectors for retinal gene delivery. Currently utilized vectors have relatively slow onset and for efficient transduction it is necessary to deliver treatment subretinally, with the potential for damage to the retina. Amino-acid substitutions in the viral capsid improve efficiency in rodent eyes by evading host responses. As dogs are important large animal models for human retinitis pigmentosa, we evaluated the speed and efficiency of retinal transduction using capsid-mutant vectors injected both subretinally and intravitreally. We evaluated AAV serotypes 2 and 8 with amino-acid substitutions of surface exposed capsid tyrosine residues. The chicken beta-actin promoter was used to drive green fluorescent protein expression. Twelve normal adult beagles were injected, 4 dogs received intravitreal injections, 8 dogs received subretinal injections. Capsid-mutant viruses tested included AAV2(quad Y-F) (intravitreal and subretinal), and self-complementary scAAV8(Y733F) (subretinal only). Contralateral control eyes received injections of scAAV5 (subretinal) or scAAV2 (intravitreal). Subretinally delivered vectors had a faster expression onset than intravitreally delivered vectors. Subretinally delivered scAAV8(Y733F) had a faster onset of expression than scAAV5. All subretinally injected vector types transduced the outer retina with high efficiency, and the inner retina with moderate efficiency. Intravitreally delivered AAV2(quad Y-F) had a marginally higher efficiency of transduction of both outer retinal and inner retinal cells than scAAV2. Because of their rapid expression onset and efficient transduction, subretinally delivered capsid-mutant AAV8 vectors may increase the efficacy of gene therapy treatment for rapid photoreceptor degenerative diseases. With further refinement, capsid-mutant AAV2 vectors show promise for retinal gene delivery from an intravitreal approach.
doi:10.1038/gt.2013.64
PMCID: PMC3880610  PMID: 24225638
(3–6): gene therapy; rAAV; canine; retina; capsid-mutant
4.  Quantifying transduction efficiencies of unmodified and tyrosine capsid mutant AAV vectors in vitro using two ocular cell lines 
Molecular Vision  2011;17:1090-1102.
Purpose
With the increasing number of retinal gene-based therapies and therapeutic constructs, in vitro bioassays characterizing vector transduction efficiency and quality are becoming increasingly important. Currently, in vitro assays quantifying vector transduction efficiency are performed predominantly for non-ocular tissues. A human retinal pigment epithelial cell line (ARPE19) and a mouse cone photoreceptor cell line, 661W, have been well characterized and are used for many retinal metabolism and biologic pathway studies. The purpose of this study is to quantify transduction efficiencies of a variety of self-complementary (sc) adeno-associated virus (AAV) vectors in these biologically relevant ocular cell lines using high-throughput fluorescence-activated cell sorting (FACS) analysis.
Methods
ARPE19 and 661W cells were infected with sc-smCBA-mCherry packaged in unmodified AAV capsids or capsids containing single/multiple tyrosine-phenylalanine (Y-F) mutations at multiplicity of infections (MOIs) ranging from 100 to 10,000. Three days post infection fluorescent images verified mCherry expression. Following microscopy, FACS analysis was performed to quantify the number of positive cells and the mean intensity of mCherry fluorescence, the product of which is reported as transduction efficiency for each vector. The scAAV vectors containing cone-specific (sc-mCARpro-green fluorescent protein [GFP]), rod-specific (sc-MOPS500-eGFP), retinal pigment epithelium (RPE)-specific (sc-VMD2-GFP), or ubiquitous (sc-smCBA-GFP) promoters were used to infect both cell lines at an MOI of 10,000. Three days post infection, cells were immunostained with an antibody raised against GFP and imaged. Finally, based on our in vitro results, we tested a prediction of transduction efficiency in vivo.
Results
Expression from unmodified scAAV1, scAAV2, scAAV5, and scAAV8 vectors was detectable by FACS in both ARPE19 and 661W cells, with scAAV1 and scAAV2 being the most efficient in both cell lines. scAAV5 showed moderate efficiency in both ARPE19 and 661W cells. scAAV8 was moderately efficient in 661W cells and was by comparison less so in ARPE19 cells; however, transduction was still apparent. scAAV9 performed poorly in both cell types. With some exceptions, the Y-F capsid mutations generally increased the efficiency of scAAV vector transduction, with the increasing number of mutated residues improving efficiency. Results for single scAAV1 and scAAV8 capsid mutants were mixed. In some cases, efficiency improved; in others, it was unchanged or marginally reduced. Retinal-specific promoters were also active in both cell lines, with the 661W cells showing a pattern consistent with the in vivo activity of the respective promoters tested. The prediction based on in vitro data that AAV2 sextuple Y-F mutants would show higher transduction efficiency in RPE relative to AAV2 triple Y-F capsid mutants was validated by evaluating the transduction characteristics of the two mutant vectors in mouse retina.
Conclusions
Our results suggest that this rapid and quantifiable cell-based assay using two biologically relevant ocular cell lines will prove useful in screening and optimizing AAV vectors for application in retina-targeted gene therapies.
PMCID: PMC3087449  PMID: 21552473
5.  Transduction of the inner mouse retina using AAVrh8 and AAVrh10 viaintravitreal injection 
Experimental eye research  2010;91(5):652-659.
Adeno-associated virus (AAV) is a proven, safe and effective vector for gene delivery in the retina. There are over 100 serotypes of AAV, and AAV2 through AAV9 have been evaluated in the retina. Each AAV serotype has different cell tropism and transduction efficiency. Intravitreal injections of AAV into the eye tend to transduce cells in the ganglion cell layer (GCL), while subretinal injections tend to transduce retinal pigment epithelium and photoreceptors. Efficient transduction of the inner retina beyond the GCL is not well established with the current methodologies and serotypes used to date. In this study, we compared the cellular tropism of AAVrh8 and AAVrh10 vectors encoding enhanced green fluorescent protein (EGFP) using intravitreal injections. We found that AAVrh8 largely transduced cells in the GCL and also amacrine cells in the inner nuclear layer (INL), as well as Müller and horizontal cells. Inner retinal transduction with AAVrh10 was similar to AAVrh8, but AAVrh10 appeared to also transduce bipolar cells. The transduction efficiency as measured by the intensity of EGFP signal was 3.5 fold higher in horizontal cells transduced with AAVrh10 than AAVrh8. Glial fibrillary accessory protein (GFAP) levels were increased in Müller cells in transduced areas for both serotypes. The results of this study suggest that AAVrh8 and AAVrh10 may be excellent vector candidates to deliver genetic material to the INL, particularly for amacrine and horizontal cells, however they may also cause cellular stress as shown by increased glial GFAP expression.
doi:10.1016/j.exer.2010.08.011
PMCID: PMC2962726  PMID: 20723541
Adeno-associated virus; AAV; inner retina; inner nuclear layer; horizontal cells; retina; retinal gene delivery
6.  Gene therapy following subretinal AAV5 vector delivery is not affected by a previous intravitreal AAV5 vector administration in the partner eye 
Molecular Vision  2009;15:267-275.
Purpose
In an earlier study we found normal adeno-associated viral vector type 2 (AAV2)-mediated GFP expression after intravitreal injection to one eye of normal C57BL/6J mice. However, GFP expression was very poor in the partner eye of the same mouse if this eye received an intravitreal injection of the same vector one month after the initial intravitreal injection. We also found both injections worked well if they were subretinal. In this study, we tested whether the efficiency of subretinal AAV vector transduction is altered by a previous intravitreal injection in the partner eye and more importantly whether therapeutic efficiency is altered in the rd12 mouse (with a recessive RPE65 mutation) after the same injection series.
Methods
One μl of scAAV5-smCBA-GFP (1x1013 genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old C57BL/6J mice and 1 μl of scAAV5-smCBA-hRPE65 (1x1013 genome containing viral particles per ml) was intravitreally injected into the right eyes of four-week-old rd12 mice Four weeks later, the same vectors were subretinally injected into the left eyes of the same C57BL/6J and rd12 mice. Left eyes of another cohort of eight-week-old rd12 mice received a single subretinal injection of the same scAAV5-smCBA-hRPE65 vector as the positive control. Dark-adapted electroretinograms (ERGs) were recorded five months after the subretinal injections. AAV-mediated GFP expression in C57BL/6J mice and RPE65 expression and ERG restoration in rd12 mice were evaluated five months after the second subretinal injection. Frozen section analysis was performed for GFP fluorescence in C57BL/6J mice and immunostaining for RPE65 in rd12 eyes.
Results
In rd12 mice, dark-adapted ERGs were minimal following the first intravitreal injection of scAAV5-smCBA-RPE65. Following subsequent subretinal injection in the partner eye, dramatic ERG restoration was recorded in that eye. In fact, ERG b-wave amplitudes were statistically similar to those from the eyes that received the initial subretinal injection at a similar age. In C57BL/6J mice, GFP positive cells were detected in eyes following the first intravitreal injection around the injection site. Strong GFP expression in both the retinal pigment epithelium (RPE) and photoreceptor (PR) cells was detected in the partner eyes following the subsequent subretinal injection. Immunostaining of retinal sections with anti-RPE65 antibody showed strong RPE65 expression mainly in the RPE cells of subretinally injected eyes but not in the intravitreally injected eyes except minimally around the injection site.
Conclusions
These results show that an initial intravitreal injection of AAV vectors to one eye of a mouse does not influence AAV-mediated gene expression or related therapeutic effects in the other eye when vectors are administered to the subretinal space. This suggests that the subretinal space possesses a unique immune privilege relative to the vitreous cavity.
PMCID: PMC2633462  PMID: 19190735
7.  A Single Intravenous AAV9 Injection Mediates Bilateral Gene Transfer to the Adult Mouse Retina 
PLoS ONE  2013;8(4):e61618.
Widespread gene delivery to the retina is an important challenge for the treatment of retinal diseases, such as retinal dystrophies. We and others have recently shown that the intravenous injection of a self-complementary (sc) AAV9 vector can direct efficient cell transduction in the central nervous system, in both neonatal and adult animals. We show here that the intravenous injection of scAAV9 encoding green fluorescent protein (GFP) resulted in gene transfer to all layers of the retina in adult mice, despite the presence of a mature blood-eye barrier. Cell morphology studies and double-labeling with retinal cell-specific markers showed that GFP was expressed in retinal pigment epithelium cells, photoreceptors, bipolar cells, Müller cells and retinal ganglion cells. The cells on the inner side of the retina, including retinal ganglion cells in particular, were transduced with the highest efficiency. Quantification of the cell population co-expressing GFP and Brn-3a showed that 45% of the retinal ganglion cells were efficiently transduced after intravenous scAAV9-GFP injection in adult mice. This study provides the first demonstration that a single intravenous scAAV9 injection can deliver transgenes to the retinas of both eyes in adult mice, suggesting that this vector serotype is able to cross mature blood-eye barriers. This intravascular gene transfer approach, by eliminating the potential invasiveness of ocular surgery, could constitute an alternative when fragility of the retina precludes subretinal or intravitreal injections of viral vectors, opening up new possibilities for gene therapy for retinal diseases.
doi:10.1371/journal.pone.0061618
PMCID: PMC3626698  PMID: 23613884
8.  Efficient expression of self-complementary AAV in ganglion cells of the ex vivo primate retina 
Molecular Vision  2009;15:2796-2802.
Purpose
To evaluate the efficiency of self-complementary adeno-associated virus (scAAV)-mediated gene expression of green fluorescent protein (GFP) or the allotopic human ND4 subunit of complex I in ganglion cells of the primate retina.
Methods
ScAAV2 containing the cDNA encoding the humanized GFP or allotopic ND4 subunit of complex I under the control of the cytomegalovirus (CMV) immediate early gene enhancer and short chicken beta-actin promoter-exon1-intron1 (CBA) was injected into the vitreous cavity of five primate eyes after enucleation. Following incubation in standard Dulbecco's Modified Eagle Medium (DMEM) culture media overnight at 37 °C with 5% CO2, retinal flat mounts were probed with monoclonal GFP or FLAG antibodies overnight followed by counterstaining with anti-mouse IgG conjugated to cy2. For identification of retinal ganglion cells (RGCs), the retinal whole mounts were also stained with a Brn3a or Thy1.2 (protein expressed in RGCs. domain) antibody, then counterstained with cy3 or cy2. Immunofluorescence and colocalization were assessed using confocal microscopy. Quantitative analysis of GFP, ND4FLAG, Brn3a, or Thy1.2 expressing cells was performed using Image J software.
Results
While the endogenous fluorescence of GFP was seen in a few retinal cells, GFP and ND4FLAG immunofluorescence was plentiful. The immunosignals were restricted to the inner retina and colocalized to slightly more than half of all cells expressing Brn3a or Thy1.2, suggesting efficient expression in RGCs.
Conclusions
Our findings suggest that the hybrid CMV enhancer-CΒA promoter can play an efficient role in targeting primate RGCs following intravitreal gene delivery using the scAAV2 vector. Donated ex vivo primate eyes may serve as a model system for testing RGC expression before in vivo intravitreal injections of this and perhaps other AAV serotypes.
PMCID: PMC2793898  PMID: 20019878
9.  Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders 
PLoS ONE  2013;8(1):e54146.
Background
Adeno-associated virus (AAV) is well established as a vehicle for in vivo gene transfer into the mammalian retina. This virus is promising not only for gene therapy of retinal diseases, but also for in vivo functional analysis of retinal genes. Previous reports have shown that AAV can infect various cell types in the developing mouse retina. However, AAV tropism in the developing retina has not yet been examined in detail.
Methodology/Principal Findings
We subretinally delivered seven AAV serotypes (AAV2/1, 2/2, 2/5, 2/8, 2/9, 2/10, and 2/11) of AAV-CAG-mCherry into P0 mouse retinas, and quantitatively evaluated the tropisms of each serotype by its infecting degree in retinal cells. After subretinal injection of AAV into postnatal day 0 (P0) mouse retinas, various retinal cell types were efficiently transduced with different AAVs. Photoreceptor cells were efficiently transduced with AAV2/5. Retinal cells, except for bipolar and Müller glial cells, were efficiently transduced with AAV2/9. Horizontal and/or ganglion cells were efficiently transduced with AAV2/1, AAV2/2, AAV2/8, AAV2/9 and AAV2/10. To confirm the usefulness of AAV-mediated gene transfer into the P0 mouse retina, we performed AAV-mediated rescue of the Cone-rod homeobox gene knockout (Crx KO) mouse, which exhibits an outer segment formation defect, flat electroretinogram (ERG) responses, and photoreceptor degeneration. We injected an AAV expressing Crx under the control of the Crx 2kb promoter into the neonatal Crx KO retina. We showed that AAV mediated-Crx expression significantly decreased the abnormalities of the Crx KO retina.
Conclusion/Significance
In the current study, we report suitable AAV tropisms for delivery into the developing mouse retina. Using AAV2/5 in photoreceptor cells, we demonstrated the possibility of gene replacement for the developmental disorder and subsequent degeneration of retinal photoreceptors caused by the absence of Crx.
doi:10.1371/journal.pone.0054146
PMCID: PMC3545928  PMID: 23335994
10.  Glycosidic enzymes enhance retinal transduction following intravitreal delivery of AAV2 
Molecular Vision  2011;17:1771-1783.
Purpose
To determine whether the co-injection of extracellular matrix degrading enzymes improves retinal transduction following intravitreal delivery of adeno-associated virus-2 (AAV2).
Methods
AAV2 containing cDNA encoding enhanced green fluorescent protein (GFP), under the control of a chicken β-actin promoter, was delivered by intravitreal injection to adult mice in conjunction with enzymes including collagenase, hyaluronan lyase, heparinase III, or chondroitin ABC lyase. Two weeks later, retinal flatmounts were examined for GFP expression using confocal microscopy.
Results
Without the addition of enzymes, transduction was limited to occasional cells in the retinal ganglion cell layer. The addition of heparinase III or chondroitin ABC lyase greatly enhanced transduction of the retinal ganglion cell layer and increased the depth of transduction into the outer retina. Hyaluronan lyase had a limited effect and collagenase was ineffective. Electroretinograms survived with higher concentrations of heparinase III and chondroitin ABC lyase than were required for optimal retinal transduction.
Conclusions
AAV2-mediated retinal transduction is improved by co-injection of heparinase III or chondroitin ABC lyase. Improved transduction efficiency may allow intravitreal injection to become the preferred route for delivering gene therapy to both the inner and outer retina.
PMCID: PMC3133842  PMID: 21750604
11.  Evaluation of AAV-Mediated Expression of Chop2-GFP in the Marmoset Retina 
This study reports for the first time the expression of channelopsin-GFP in inner retinal neurons of a nonhuman primate, the common marmoset. The study constitutes an important step in developing a channelrhodopsin-2–based gene therapy for treating blinding retinal degenerative diseases in humans.
Purpose.
Converting inner retinal neurons to photosensitive cells by expressing channelrhodopsin-2 (ChR2) offers a novel approach for treating blindness caused by retinal degenerative diseases. In the present study, the recombinant adeno-associated virus serotype 2 (rAAV2)–mediated expression and function of a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP) (Chop2-GFP) were evaluated in the inner retinal neurons in the common marmoset Callithrix jacchus.
Methods.
rAAV2 vectors carrying ubiquitous promoters were injected into the vitreous chamber. Expression of Chop2-GFP and functional properties of ChR2 were examined by immunocytochemical and electrophysiological methods 3 months after injection.
Results.
The percentage of Chop2-GFP–expressing cells in the ganglion cell layer was found to be retinal region- and animal age-dependent. The highest percentage was observed in the far-peripheral region. Chop2-GFP expression was also found in the foveal and parafoveal region. In the peripheral retina in young animals with high viral concentrations, the expression of Chop2-GFP was observed in all major classes of retinal neurons, including all major types of ganglion cells. The morphologic properties of Chop2-GFP–positive cells were normal for at least 3 months, and ChR2-mediated light responses were demonstrated by electrophysiological recordings.
Conclusions.
The rAAV2-mediated expression of ChR2 was observed in the inner retinal neurons in the marmoset retina through intravitreal delivery. The marmoset could be a valuable nonhuman primate model for developing ChR2-based gene therapy for treating blinding retinal degenerative diseases.
doi:10.1167/iovs.10-5389
PMCID: PMC2939198  PMID: 20484599
12.  Virus-Mediated Transduction of Murine Retina with Adeno-Associated Virus: Effects of Viral Capsid and Genome Size 
Journal of Virology  2002;76(15):7651-7660.
Gene therapy vectors based on adeno-associated viruses (AAVs) show promise for the treatment of retinal degenerative diseases. In prior work, subretinal injections of AAV2, AAV5, and AAV2 pseudotyped with AAV5 capsids (AAV2/5) showed variable retinal pigmented epithelium (RPE) and photoreceptor cell transduction, while AAV2/1 predominantly transduced the RPE. To more thoroughly compare the efficiencies of gene transfer of AAV2, AAV3, AAV5, and AAV6, we quantified, using stereological methods, the kinetics and efficiency of AAV transduction to mouse photoreceptor cells. We observed persistent photoreceptor and RPE transduction by AAV5 and AAV2 up to 31 weeks and found that AAV5 transduced a greater volume than AAV2. AAV5 containing full-length or half-length genomes and AAV2/5 transduced comparable numbers of photoreceptor cells with similar rates of onset of expression. Compared to AAV2, AAV5 transduced significantly greater numbers of photoreceptor cells at 5 and 15 weeks after surgery (greater than 1,000 times and up to 400 times more, respectively). Also, there were 30 times more genome copies in eyes injected with AAV2/5 than in eyes injected with AAV2. Comparing AAVs with half-length genomes, AAV5 transduced only four times more photoreceptor cells than AAV2 at 5 weeks and nearly equivalent numbers at 15 weeks. The enhancement of transduction was seen at the DNA level, with 50 times more viral genome copies in retinas injected with AAV having short genomes than in retinas injected with AAV containing full-length ones. Subretinal injection of AAV2/6 showed only RPE transduction at 5 and 15 weeks, while AAV2/3 did not transduce retinal cells. We conclude that varying genome length and AAV capsids may allow for improved expression and/or gene transfer to specific cell types in the retina.
doi:10.1128/JVI.76.15.7651-7660.2002
PMCID: PMC136354  PMID: 12097579
13.  Evaluation of AAV-Mediated Expression of Chop2-GFP in the Marmoset Retina 
Purpose
Converting inner retinal neurons to photosensitive cells by expressing channelrhodopsin-2 (ChR2) offers a novel approach for treating blindness caused by retinal degenerative diseases. We evaluated the recombinant adeno-associated virus serotype 2 (rAAV2)-mediated expression and function of a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP) (Chop2-GFP) in inner retinal neurons in the common marmoset Callithrix jacchus.
Methods
rAAV2 vectors carrying ubiquitous promoters were injected into the vitreous chamber. Expression of Chop2-GFP and functional properties of ChR2 were examined 3 months after injection with immunocytochemical and electrophysiological methods.
Results
The percentage of Chop2-GFP-expressing cells in the ganglion cell layer was found to be retinal region- and animal age-dependent. The highest percentage was observed in the far-peripheral region. Chop2-GFP expression was also found in foveal and para-foveal region. In the peripheral retina in young animals with high viral concentrations, the expression of Chop2-GFP was observed in all major classes of retinal neurons, including all major types of ganglion cells. The morphological properties of Chop2-GFP-positive cells were normal for at least three months; and ChR2-mediated light responses were demonstrated by electrophysiological recordings.
Conclusions
We reported the rAAV2-mediated expression of ChR2 in the inner retinal neurons in the marmoset retina through intravitreal delivery. The marmoset could be a valuable non-human primate model for developing ChR2-based gene therapy for treating blinding retinal degenerative diseases.
doi:10.1167/iovs.10-5389
PMCID: PMC2939198  PMID: 20484599
14.  Functional expression of Rab escort protein 1 following AAV2-mediated gene delivery in the retina of choroideremia mice and human cells ex vivo 
Choroideremia (CHM) is an X-linked retinal degeneration of photoreceptors, the retinal pigment epithelium (RPE) and choroid caused by loss of function mutations in the CHM/REP1 gene that encodes Rab escort protein 1. As a slowly progressing monogenic retinal degeneration with a clearly identifiable phenotype and a reliable diagnosis, CHM is an ideal candidate for gene therapy. We developed a serotype 2 adeno-associated viral vector AAV2/2-CBA-REP1, which expresses REP1 under control of CMV-enhanced chicken β-actin promoter (CBA) augmented by a Woodchuck hepatitis virus post-transcriptional regulatory element. We show that the AAV2/2-CBA-REP1 vector provides strong and functional transgene expression in the D17 dog osteosarcoma cell line, CHM patient fibroblasts and CHM mouse RPE cells in vitro and in vivo. The ability to transduce human photoreceptors highly effectively with this expression cassette was confirmed in AAV2/2-CBA-GFP transduced human retinal explants ex vivo. Electroretinogram (ERG) analysis of AAV2/2-CBA-REP1 and AAV2/2-CBA-GFP-injected wild-type mouse eyes did not show toxic effects resulting from REP1 overexpression. Subretinal injections of AAV2/2-CBA-REP1 into CHM mouse retinas led to a significant increase in a- and b-wave of ERG responses in comparison to sham-injected eyes confirming that AAV2/2-CBA-REP1 is a promising vector suitable for choroideremia gene therapy in human clinical trials.
Electronic supplementary material
The online version of this article (doi:10.1007/s00109-013-1006-4) contains supplementary material, which is available to authorized users.
doi:10.1007/s00109-013-1006-4
PMCID: PMC3695676  PMID: 23756766
Rab escort protein 1; Gene therapy; Choroideremia; Rab GTPase; Retinitis pigmentosa; AAV
15.  Self-complementary AAV Virus (scAAV) Safe and Long-term Gene Transfer in the Trabecular Meshwork of Living Rats and Monkeys 
In this paper, the authors identify a modified adeno-associated viral (AAV) vector, self-complementary AAV (scAAV) that, in contrast to the conventional AAV, transduces the trabecular meshwork of living rats and monkeys. scAAV confers long-term expression to the trabecular meshwork (>3.5 months in rats and >2.35 years in monkeys) without clinical adverse effects.
Purpose.
AAV vectors produce stable transgene expression and elicit low immune response in many tissues. AAVs have been the vectors of choice for gene therapy for the eye, in particular the retina. scAAVs are modified AAVs that bypass the required second-strand DNA synthesis to achieve transcription of the transgene. The goal was to investigate the ability of AAV vectors to induce long-term, safe delivery of transgenes to the trabecular meshwork of living animals.
Methods.
Single doses of AAV2.GFP and AAV2.RGD.GFP/Ad5.LacZ were injected intracamerally (IC) into rats (n = 28 eyes). A single dose of scAAV.GFP was IC-injected into rats (n = 72 eyes) and cynomolgus monkeys (n = 3). GFP expression was evaluated by fluorescence, immunohistochemistry, and noninvasive gonioscopy. Intraocular pressure (IOP) was measured with calibrated tonometer (rats) and Goldmann tonometer (monkeys). Differential expression of scAAV-infected human trabecular meshwork cells (HTM) was determined by microarrays. Humoral and cell-mediated immune responses were evaluated by ELISA and peripheral blood proliferation assays.
Results.
No GFP transduction was observed on the anterior segment tissues of AAV-injected rats up to 27 days after injection. In contrast, scAAV2 transduced the trabecular meshwork very efficiently, with a fast onset (4 days). Eyes remained clear and no adverse effects were observed. Transgene expression lasted >3.5 months in rats and >2.35 years in monkeys.
Conclusions.
The scAAV viral vector provides prolonged and safe transduction in the trabecular meshwork of rats and monkeys. The stable expression and safe properties of this vector could facilitate the development of trabecular meshwork drugs for gene therapy for glaucoma.
doi:10.1167/iovs.09-3847
PMCID: PMC2869048  PMID: 19684004
16.  Adeno-associated virus serotype-9 efficiently transduces the retinal outer plexiform layer 
Molecular Vision  2009;15:1374-1382.
Purpose
Adeno-associated virus serotype-9 (AAV-9) is a promising gene delivery vector. In this study, we evaluated AAV-9 transduction in the mouse retina.
Methods
Three different AAV vectors were used in our study: AAV-9.RSV.AP, AAV-9.CMV.eGFP, and AAV-9.CMV.∆R4–23/∆C. In these vectors, two different promoters (the cytomegalovirus promoter-CMV promoter and the Rous sarcoma virus-RSV promoter) were used to express three different transgenes including the alkaline phosphatase (AP) gene, the enhanced green fluorescent protein (eGFP) gene, and a therapeutic microdystrophin gene (the ∆R4–23/∆C). Specifically, 1 µl AAV-9 reporter gene vectors (1×109 viral genome particles of AAV-9.RSV.AP or 1×1010 viral genome particles of AAV-9.CMV.eGFP) were administered subretinally to young (2–3-week-old), adult (3-month-old), and old (12-month-old) C57BL/6J mice. To evaluate AAV-9 transduction in a diseased retina, we injected subretinally 1×109 viral genome particles of AAV-9.CMV.∆R4–23/∆C to mdx3cv mice, which we used as a model for Duchenne muscular dystrophy (DMD). Transgene expression was examined by histochemical as well as immunofluorescence staining at three and five weeks after injection. Electroretinograms were recorded five weeks after subretinal AAV-9.RSV.AP injection.
Results
Subretinal injection yielded widespread transduction throughout the retina in all age groups. Robust expression was seen in the retinal pigment epithelium, outer nuclear layer, and in Müller cells. Interestingly a synaptic layer, the outer plexiform layer (OPL), also showed intensive expression. Transduction of the synaptic layer was further confirmed by immunostaining for C-terminal binding protein 2 (CtBP2), a marker for the photoreceptor synaptic ribbon. Dystrophin is normally expressed in the OPL photoreceptor terminals. This expression is lost in DMD patients and mdx3cv mice. Consistent with our findings in normal mice, we observed efficient microdystrophin expression in the OPL after AAV-9.CMV.∆R4–23/∆C infection. At five weeks after subretinal delivery of AAV-9.RSV.AP, no morphology or ERG abnormalities were observed.
Conclusions
We demonstrated that AAV-9 is a potent vector for retinal gene delivery. Furthermore, subretinal AAV-9 administration did not cause appreciable acute retinal damages. In summary, AAV-9-mediated OPL transduction holds promise for treating diseases that primarily affect this layer.
PMCID: PMC2713732  PMID: 19626133
17.  Mutant NADH dehydrogenase subunit 4 gene delivery to mitochondria by targeting sequence-modified adeno-associated virus induces visual loss and optic atrophy in mice 
Molecular Vision  2012;18:1668-1683.
Purpose
Although mutated G11778A NADH ubiquinone oxidoreductase subunit 4 (ND4) mitochondrial DNA (mtDNA) is firmly linked to the blindness of Leber hereditary optic neuropathy (LHON), a bona fide animal model system with mutated mtDNA complex I subunits that would enable probing the pathogenesis of optic neuropathy and testing potential avenues for therapy has yet to be developed.
Methods
The mutant human ND4 gene with a guanine to adenine transition at position 11778 with an attached FLAG epitope under control of the mitochondrial heavy strand promoter (HSP) was inserted into a modified self-complementary (sc) adeno-associated virus (AAV) backbone. The HSP-ND4FLAG was directed toward the mitochondria by adding the 23 amino acid cytochrome oxidase subunit 8 (COX8) presequence fused in frame to the N-terminus of green fluorescent protein (GFP) into the AAV2 capsid open reading frame. The packaged scAAV-HSP mutant ND4 was injected into the vitreous cavity of normal mice (OD). Contralateral eyes received scAAV-GFP (OS). Translocation and integration of mutant human ND4 in mouse mitochondria were assessed with PCR, reverse transcription–polymerase chain reaction (RT–PCR), sequencing, immunoblotting, and immunohistochemistry. Visual function was monitored with serial pattern electroretinography (PERG) and in vivo structure with spectral domain optical coherence tomography (OCT). Animals were euthanized at 1 year and processed for light and transmission electron microscopy.
Results
The PCR products of the mitochondrial and nuclear DNA extracted from infected retinas and optic nerves gave the expected 500 base pair bands. RT–PCR confirmed transcription of the mutant human ND4 DNA in mice. DNA sequencing confirmed that the PCR and RT–PCR products were mutant human ND4 (OD only). Immunoblotting revealed the expression of mutant ND4FLAG (OD only). Pattern electroretinograms showed a significant decrement in retinal ganglion cell function OD relative to OS at 1 month and 6 months after AAV injections. Spectral domain optical coherence tomography showed optic disc edema starting at 1 month post injection followed by optic nerve head atrophy with marked thinning of the inner retina at 1 year. Histopathology of optic nerve cross sections revealed reductions in the optic nerve diameters of OD versus OS where transmission electron microscopy revealed significant loss of optic nerve axons in mutant ND4 injected eyes where some remaining axons were still in various stages of irreversible degeneration with electron dense aggregation. Electron lucent mitochondria accumulated in swollen axons where fusion of mitochondria was also evident.
Conclusions
Due to the UGA codon at amino acid 16, mutant G11778A ND4 was translated only in the mitochondria where its expression led to significant loss of visual function, loss of retinal ganglion cells, and optic nerve degeneration recapitulating the hallmarks of human LHON.
PMCID: PMC3388991  PMID: 22773905
18.  AAV retinal transduction in a large animal model species: Comparison of a self-complementary AAV2/5 with a single-stranded AAV2/5 vector 
Molecular Vision  2009;15:1835-1842.
Purpose
To compare self-complementary (sc) and single-stranded (ss) adeno-associated viral 2/5 (AAV2/5) vectors for retinal cell transduction in the dog when delivered by subretinal injection.
Methods
ScAAV2/5 and ssAAV2/5 vectors encoding enhanced green fluorescent protein (GFP) under control of the chicken beta actin promoter were prepared to the same titer. Equal amounts of viral particles were delivered into the subretinal spaces of both eyes of two dogs. In each dog, one eye received the scAAV2/5 and the other the ssAAV2/5. In vivo expression of GFP was monitored ophthalmoscopically. The dogs were sacrificed, and their retinas were examined by fluorescent microscopy and immunohistochemistry to determine GFP expression patterns and to assay for glial reactivity.
Results
GFP expression in the scAAV2/5 injected eyes was detectable at a much earlier time point than in the ssAAV2/5 injected eyes. Expression of GFP was also at higher levels in the scAAV2/5-injected eyes. Expression levels remained stable for the seven month duration of the study. The types of cells transduced by both vectors were similar; there was strong reporter gene expression in the RPE and photoreceptors, although not all cones in the transduced area expressed GFP. Some horizontal and Müller cells were also transduced.
Conclusions
When delivered by subretinal injection in the dog, scAAV2/5 induces faster and stronger transgene expression than ssAAV2/5. The spectrum of retinal neurons transduced is similar between the two vectors. These results confirm in a large animal model those previously reported in the mouse. ScAAV2/5 shows promise for use in the treatment of conditions where a rapid transgene expression is desirable. Furthermore, it may be possible to use a lower number of viral particles to achieve the same effect compared with ssAAV2/5 vectors.
PMCID: PMC2743804  PMID: 19756181
19.  Gradients of Eph-A6 expression in primate retina suggest roles in both vascular and axon guidance 
Molecular Vision  2009;15:2649-2662.
Purpose
Recently we identified high levels of expression of Eph-A6 in the macula of developing human retina and showed localization of Eph-A6 to ganglion cells (GC). In the present study we investigated the expression of some members of the ephrin family in developing primate retina, including the topography of Eph-A6 expression, and its ligands, in developing macaque retinas.
Methods
We extracted RNA from human fetal retinas and probed for Eph-A5–A7, Eph-B1, ephrin-B2, and ephrin-A1-A5 by RT–PCR, then prepared riboprobes for Eph-A5-A7, Eph-B1 and ephrin-A1, -A4 and -B2. Paraffin sections of fetal macaque retinas were used to localize expression of Ephs and ephrins by in situ hybridization and immunohistochemistry.
Results
We identified prominent gradients of Eph-A6 mRNA expression in the ganglion cell layer (GCL) of fetal macaque retinas of different ages. The gradient of Eph-A6 expression was high near the optic disc and low at the developing macula at fetal day (Fd) 55. At Fd 70 and 80, the gradient of Eph-A6 expression was reversed, being higher temporal to the macula, and low at the disc. By Fd 110, when the fovea begins to form, a pattern of expression was established that persisted into the postnatal period, in which the highest levels of expression were detected at the developing fovea, and progressively lower levels of expression were detected at increasing distance from the fovea. Beginning at Fd 70, we also detected a gradient of Eph-A6 expression running perpendicular to the retinal surface within the GCL of central retina that was high in the inner GCL and low in the outer GCL. This second pattern persisted into the neonatal period. We found the two ligands for Eph-A6, ephrin-A1 and ephrin-A4, expressed by Pax2-immunoreactive astrocytes, in the optic nerve head and in the retina, by in situ hybridization and immunohistochemistry. We propose that during development of the retinal vasculature, migration of ligand-bearing astrocytes is slowed along this Eph-A6 expression gradient through repellent Eph-A6 - ephrin-A1 and -A4 signaling.
Conclusions
Patterns of Eph-A6 expression in the developing macaque retina suggest that Eph-A6 - ephrin-A1 and -A4 repellent signaling has a role in retinal vascular patterning, and in the postnatal maintenance of projections from macular and foveal GC.
PMCID: PMC2791039  PMID: 20011078
20.  GFAP-Driven GFP Expression in Activated Mouse Müller Glial Cells Aligning Retinal Blood Vessels Following Intravitreal Injection of AAV2/6 Vectors 
PLoS ONE  2010;5(8):e12387.
Background
Müller cell gliosis occurs in various retinal pathologies regardless of the underlying cellular defect. Because activated Müller glial cells span the entire retina and align areas of injury, they are ideal targets for therapeutic strategies, including gene therapy.
Methodology/Principal Findings
We used adeno-associated viral AAV2/6 vectors to transduce mouse retinas. The transduction pattern of AAV2/6 was investigated by studying expression of the green fluorescent protein (GFP) transgene using scanning-laser ophthalmoscopy and immuno-histochemistry. AAV2/6 vectors transduced mouse Müller glial cells aligning the retinal blood vessels. However, the transduction capacity was hindered by the inner limiting membrane (ILM) and besides Müller glial cells, several other inner retinal cell types were transduced. To obtain Müller glial cell-specific transgene expression, the cytomegalovirus (CMV) promoter was replaced by the glial fibrillary acidic protein (GFAP) promoter. Specificity and activation of the GFAP promoter was tested in a mouse model for retinal gliosis. Mice deficient for Crumbs homologue 1 (CRB1) develop gliosis after light exposure. Light exposure of Crb1−/− retinas transduced with AAV2/6-GFAP-GFP induced GFP expression restricted to activated Müller glial cells aligning retinal blood vessels.
Conclusions/Significance
Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.
doi:10.1371/journal.pone.0012387
PMCID: PMC2927518  PMID: 20808778
21.  Long-term Preservation of Cone Photoreceptors and Restoration of Cone Function by Gene Therapy in the Guanylate Cyclase-1 Knockout (GC1KO) Mouse 
This study demonstrates for the first time that long-term therapy (∼1 year) is achievable in a mammalian model of GC1 deficiency. These data provide additional justification for the development of an AAV-based gene therapy vector for the clinical treatment of LCA1.
Purpose.
The authors previously showed that subretinal delivery of AAV5 vectors containing murine guanylate cyclase-1 (GC1) cDNA driven by either photoreceptor-specific (hGRK1) or ubiquitous (smCBA) promoters was capable of restoring cone-mediated function and visual behavior and preserving cone photoreceptors in the GC1 knockout (GC1KO) mouse for 3 months. Here, the authors compared therapy conferred by the aforementioned vectors to that achieved with the highly efficient capsid tyrosine mutant AAV8(Y733F) and asked whether long-term therapy is achievable in this model.
Methods.
AAV5-hGRK1-mGC1, AAV5-smCBA-mGC1, or AAV8(Y733F)-hGRK1-mGC1 was delivered subretinally to GC1KO mice between postnatal day (P)14 and P25. Retinal function was assayed by electroretinography. Localization of AAV-mediated GC1 expression and cone survival were assayed with immunohistochemistry, and the spread of vector genomes beyond the retina was quantified by PCR of optic nerve and brain tissue.
Results.
Cone function was restored with all vectors tested, with AAV8(Y733F) being the most efficient. Electroretinographic responses were clearly measurable out to 1 year after treatment. AAV-mediated expression of GC1 was found exclusively in photoreceptors out to 15 months after injection. Cones were preserved for at least 11 months after treatment. AAV5- and AAV8(733)–delivered vector genomes were recovered primarily from optic nerve of the treated eye and, in only instance, from brain (1 of 20 samples).
Conclusions.
The authors demonstrate for the first time that long-term therapy (∼1 year) is achievable in a mammalian model of GC1 deficiency. These data provide additional justification for the development of an AAV-based gene therapy vector for the clinical treatment of Leber congenital amaurosis-1.
doi:10.1167/iovs.11-7867
PMCID: PMC3207713  PMID: 21778276
22.  Different tropism of adenoviruses and adeno-associated viruses to corneal cells: implications for corneal gene therapy 
Molecular Vision  2008;14:2087-2096.
Purpose
Diseased corneas are potential targets for viral-based gene therapy to normalize (stimulate or inhibit) the expression of specific proteins. The choice of viral vectors is important to achieve optimal effect. The purpose of this study was to compare the tropism to different corneal cells of recombinant adenovirus (rAV) and recombinant adeno-associated virus (rAAV) constructs using live rabbit and organ-cultured human corneas.
Methods
rAV constructs harbored the green fluorescent protein (GFP) gene under the control of major immediate early cytomegalovirus (CMV) promoter. rAAV constructs from virus serotypes 1, 2 5, 7, and 8 had GFP under the chicken β-actin promoter and CMV enhancer. For organ culture, 16 healthy and diabetic postmortem human corneas were used. Five or fifteen μl rAV at 107 plaque forming units per 1 μl were added for 2 days to culture medium of uninjured corneas that were further cultured for 5–32 days. rAAV were added at 1.2–7.8×1010 vector genomes per cornea for 3 days to each cornea; the culture then continued for another 14–23 days. Corneal cryostat sections were examined by immunohistochemistry. Live rabbit corneas were used following excimer laser ablation of the corneal epithelium with preservation of the basal cell layer. Equal numbers of rAAV particles (2x1011 vector genomes) were applied to the cornea for 10 min. After seven days to allow for corneal healing and gene expression the animals were euthanized, the corneas were excised, and sections analyzed by immunohistochemistry.
Results
By direct fluorescence microscopy of live organ-cultured human corneas GFP signal after rAV transduction was strong in the epithelium with dose-dependent intensity. On corneal sections, GFP was seen in all epithelial layers and some endothelial cells but most keratocytes were negative. In rAAV-transduced organ-cultured human corneas GFP signal could only be detected with anti-GFP antibody immunohistochemistry. GFP was observed in the epithelium, keratocytes, and endothelium, with more pronounced basal epithelial cell staining with rAAV1 than with other serotypes. No difference in the GFP expression patterns or levels between normal and diabetic corneas was noted. The rabbit corneas showed very similar patterns of GFP distribution to human corneas. With all rAAV serotype vectors, GFP staining in the epithelium was significantly (p=0.007) higher than the background staining in non-transduced corneas, with a trend for rAAV1 and rAAV8 to produce higher staining intensities than for rAAV2, rAAV5 (p=0.03; rAAV5 versus rAAV1), and rAAV7. rAAV serotype vectors also transduced stromal and endothelial cells in rabbit corneas to a different extent.
Conclusions
rAAV appears to reach many more corneal cells than rAV, especially keratocytes, although GFP expression levels were lower compared to rAV. rAV may be more useful than rAAV for gene therapy applications requiring high protein expression levels, but rAAV may be superior for keratocyte targeting.
PMCID: PMC2584774  PMID: 19023450
23.  The Signalling Role of the αvβ5-Integrin Can Impact the Efficacy of AAV in Retinal Gene Therapy 
Pharmaceuticals  2012;5(5):447-459.
Sub-retinal injection of the common AAV2 pseudotypes frequently results in strong transduction of the retinal pigment epithelium (RPE) as well as the retina itself. This has been of benefit to date in human clinical trials using AAV, where the disease target is in the RPE. However, many mutations predisposing to retinal disease are located in the photoreceptor cells, present in the neural retina and not the RPE; in this case the sub-retinal injection route may cause an effective “loss” of therapeutic AAV to the RPE. The αvβ5 integrin receptor is highly expressed on the apical surface of the RPE, and is essential to the daily phagocytosis of the outer segment tips of photoreceptor cells. The transduction efficiency of AAV was tested in the retinas of β5−/− mice lacking this receptor and showing defects in photoreceptor outer segment phagocytosis. Following sub-retinal injection of AAV2/5-eGFP, fluorescence was found to be stronger and more widespread in the neural retina of β5−/− mice compared to wild-types with greatly reduced fluorescence in the RPE. Increased levels of the phagocytic signalling protein MFG-E8, the ligand for the αvβ5 integrin receptor, is found to have a moderate inhibitory effect on AAV transduction of the retina. However the opposite effect is found when only the integrin-binding domain of MFG-E8, the RGD (Arginine-Glycine-Aspartic acid) domain, was increased. In this case RGD enhanced AAV-mediated retinal transduction relative to RPE transduction. These results are presented for their relevance for the design of AAV-based retinal gene therapy strategies strategies targeting retinal/photoreceptor cells.
doi:10.3390/ph5050447
PMCID: PMC3763646  PMID: 24281556
adeno-associated virus; retinal gene therapy; MFG-E8; αvβ5 integrin; photoreceptor outer segments
24.  Vascular endothelial growth factor-B gene transfer exacerbates retinal and choroidal neovascularization and vasopermeability without promoting inflammation 
Molecular Vision  2011;17:492-507.
Purpose
The role of vascular endothelial growth factor (VEGF)-B in the eye is poorly understood. The present study was conducted to evaluate the effect of overexpression of VEGF-B via adeno-associated virus (AAV) gene transfer on ocular angiogenesis, inflammation, and the blood-retinal barrier (BRB).
Methods
Three recombinant AAV vectors were prepared, expressing the 167 (AAV-VEGF-B167) or 186 amino acid isoform (AAV-VEGF-B186) of VEGF-B or the green fluorescent protein (GFP) reporter gene (AAV-GFP). Approximately 1×109 viral genome copies of AAV-VEGF-B167, AAV-VEGF-B186, or AAV-GFP were intraocularly injected. The efficacy of the gene transfer was assessed by directly observing GFP, by immunohistochemistry, or by real-time PCR. A leukostasis assay using fluorescein isothiocyanate-conjugated Concanavalin A was used to evaluate inflammation. The BRB was assessed using a quantitative assay with 3H-mannitol as a tracer. Retinal neovascularization (NV) was assessed at postnatal day 17 in oxygen-induced ischemic retinopathy after intravitreal injection of AAV-VEGF-B in left eyes and AAV-GFP in right eyes at postnatal day 7. Two weeks after injection of AAV vectors, choroidal NV was generated by laser photocoagulation and assessed 2 weeks later.
Results
GFP expression was clearly demonstrated, primarily in the retinal pigment epithelium (RPE) and outer retina, 1–6 weeks after delivery. mRNA expression levels of VEGF-B167 and VEGF-B186 were 5.8 and 12 fold higher in the AAV-VEGF-B167- and AAV-VEGF-B186-treated groups, respectively. There was no evidence of an inflammatory response or vessel abnormality following injection of the vectors in normal mice; however, VEGF-B increased retinal and choroidal neovascularization. AAV-VEGF-B186, but not AAV-VEGF-B167, enhanced retinal vascular permeability.
Conclusions
VEGF-B overexpression promoted pathological retinal and choroidal NV and BRB breakdown without causing inflammation, which is associated with the progression of diabetic retinopathy and age-related macular degeneration, showing that these complications are not dependent on inflammation. VEGF-B targeting could benefit antiangiogenic therapy.
PMCID: PMC3042363  PMID: 21364963
25.  Recombinant Vectors Based on Porcine Adeno-Associated Viral Serotypes Transduce the Murine and Pig Retina 
PLoS ONE  2013;8(3):e59025.
Recombinant adeno-associated viral (AAV) vectors are known to safely and efficiently transduce the retina. Among the various AAV serotypes available, AAV2/5 and 2/8 are the most effective for gene transfer to photoreceptors (PR), which are the most relevant targets for gene therapy of inherited retinal degenerations. However, the search for novel AAV serotypes with improved PR transduction is ongoing. In this work we tested vectors derived from five AAV serotypes isolated from porcine tissues (referred to as porcine AAVs, four of which are newly identified) for their ability to transduce both the murine and the cone-enriched pig retina. Porcine AAV vectors expressing EGFP under the control of the CMV promoter were injected subretinally either in C57BL/6 mice or Large White pigs. The resulting retinal tropism was analyzed one month later on histological sections, while levels of PR transduction were assessed by Western blot. Our results show that all porcine AAV transduce murine and porcine retinal pigment epithelium and PR upon subretinal administration. AAV2/po1 and 2/po5 are the most efficient porcine AAVs for murine PR transduction and exhibit the strongest tropism for pig cone PR. The levels of PR transduction obtained with AAV2/po1 and 2/po5 are similar, albeit not superior, to those obtained with AAV2/5 and AAV2/8, which evinces AAV2/po1 and 2/po5 to be promising vectors for retinal gene therapy.
doi:10.1371/journal.pone.0059025
PMCID: PMC3592811  PMID: 23520549

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