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1.  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.
PMCID: PMC2962726  PMID: 20723541
Adeno-associated virus; AAV; inner retina; inner nuclear layer; horizontal cells; retina; retinal gene delivery
2.  AAV9 Targets Cone Photoreceptors in the Nonhuman Primate Retina 
PLoS ONE  2013;8(1):e53463.
Transduction of retinal pigment epithelial cells with an adeno-associated viral vector (AAV) based on serotype 2 has partially corrected retinal blindness in Leber congenital amaurosis type 2. However, many applications of gene therapy for retinal blindness rely on the efficient transduction of rod and cone photoreceptor which is difficult to achieve with first generation vector technology. To address this translational need, we evaluated rod and cone photoreceptor targeting of 4 novel AAV capsids (AAV7, AAV9, rh.64R1 and rh.8R) versus AAV2 and AAV8 in a foveated retina. Eyes of 20 nonhuman primates were injected subretinally in the proximity of the fovea. While numerous vectors efficiently transduced rods, only AAV9 targeted cones both centrally and peripherally efficiently at low doses, likely due to the abundance of galactosylated glycans, the primary receptor for AAV9, on cone photoreceptors. We conclude AAV9 is an ideal candidate for strategies that require restoration of cone photoreceptor function.
PMCID: PMC3559681  PMID: 23382846
3.  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.
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.
Our experiments indicate that AAV2 vectors carrying the GFAP promoter are a promising tool for specific expression of transgenes in activated glial cells.
PMCID: PMC2927518  PMID: 20808778
4.  Intravitreal delivery of AAV8 retinoschisin results in cell type-specific gene expression and retinal rescue in the Rs1-KO mouse 
Gene therapy  2009;16(7):916-926.
X-linked juvenile retinoschisis (XLRS) is a neurodevelopmental abnormality caused by retinoschisin gene mutations. XLRS is characterized by splitting through the retinal layers and impaired synaptic transmission of visual signals resulting in impaired acuity and a propensity to retinal detachment. Several groups have treated murine retinoschisis models successfully using adeno-associated virus (AAV) vectors. Owing to the fragile nature of XLRS retina, translating this therapy to the clinic may require an alternative to invasive subretinal vector administration. Here we show that all layers of the retinoschisin knockout (Rs1-KO) mouse retina can be transduced efficiently with AAV vectors administered by simple vitreous injection. Retinoschisin expression was restricted to the neuroretina using a new vector that uses a 3.5-kb human retinoschisin promoter and an AAV type 8 capsid. Intravitreal administration to Rs1-KO mice resulted in robust retinoschisin expression with a retinal distribution similar to that observed in wild-type retina, including the expression by the photoreceptors lying deep in the retina. No off-target expression was observed. Rs1-KO mice treated with this vector showed a decrease in the schisis cavities and had improved retinal signaling evaluated by recording the electroretinogram 11–15 weeks after the application.
PMCID: PMC2774250  PMID: 19458650
retinoschisis; AAV vector; promoter; intravitreal injection
5.  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.
PMCID: PMC3592811  PMID: 23520549
6.  Changes in Adeno-Associated Virus-Mediated Gene Delivery in Retinal Degeneration 
Human Gene Therapy  2010;21(5):571-578.
Gene therapies for retinal degeneration have relied on subretinal delivery of viral vectors carrying therapeutic DNA. The subretinal injection is clearly not ideal as it limits the viral transduction profile to a focal region at the injection site and negatively affects the neural retina by detaching it from the supportive retinal pigment epithelium (RPE). We assessed changes in adeno-associated virus (AAV) dispersion and transduction in the degenerating rat retina after intravitreal delivery. We observed a significant increase in AAV-mediated gene transfer in the diseased compared with normal retina, the extent of which depends on the AAV serotype injected. We also identified key structural changes that correspond to increased viral infectivity. Particle diffusion and transgene accumulation in normal and diseased retina were monitored via fluorescent labeling of viral capsids and quantitative PCR. Viral particles were observed to accumulate at the vitreoretinal junction in normal retina, whereas particles spread into the outer retina and RPE in degenerated tissue. Immunohistochemistry illustrates remarkable changes in the architecture of the inner limiting membrane, which are likely to underlie the increased viral transduction in diseased retina. These data highlight the importance of characterizing gene delivery vectors in diseased tissue as structural and biochemical changes can alter viral vector transduction patterns. Furthermore, these results indicate that gene delivery to the outer nuclear layer may be achieved by noninvasive intravitreal AAV administration in the diseased state.
Kolstad et al. evaluate the distribution of vector particles and transduction of AAV administered intravitreally in diseased versus healthy retinas. Whereas healthy retinas are not very receptive to vector penetration and transduction following intravitreal injection, in retinal degenerations the authors show improved and more extensive gene transfer.
PMCID: PMC3143418  PMID: 20021232
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.
PMCID: PMC3626698  PMID: 23613884
8.  Glycosidic enzymes enhance retinal transduction following intravitreal delivery of AAV2 
Molecular Vision  2011;17:1771-1783.
To determine whether the co-injection of extracellular matrix degrading enzymes improves retinal transduction following intravitreal delivery of adeno-associated virus-2 (AAV2).
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.
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.
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
9.  Evaluation of the adeno-associated virus mediated long-term expression of channelrhodopsin-2 in the mouse retina 
Molecular Vision  2009;15:1680-1689.
The conversion of inner retinal neurons to photosensitive cells via viral mediated expression of channelrhodopsin-2 (ChR2) offers a new potential approach for the restoration of vision after photoreceptor degeneration. This study was conducted to evaluate the recombinant adeno-associated virus serotype 2 (rAAV2)-mediated long-term expression and safety of ChR2 in the mouse retina.
rAAV2 vectors carrying a fusion construct of channelopsin-2 (Chop2) and green fluorescent protein (GFP; Chop2-GFP) under the control of a hybrid cytomegalovirus early enhancer and chicken β-actin (CAG) promoter were injected at different concentrations into the eyes of wild-type adult mice. The retinas were harvested up to 18 months after virus injection for immunostaining and electrophysiological studies. Injected mice were kept either under normal light conditions, or exposed to a strong blue light. The expression of GFP and the density of the cells in the ganglion cell layer (GCL) were examined.
The expression of Chop2-GFP was stable for up to 18 months. Chop-GFP was observed predominantly in retinal ganglion as well as amacrine cells. At the highest virus concentration (6×1012 GC/ml), up to 20% of the cells in the GCL were infected by the virus. At the lowest virus concentration (1×1010 GC/ml), the expression was targeted to AII amacrine cells. The concentration of the virus, the light conditions, and the percentage of Chop2-GFP-positive cells had no effect on the density and, thus, on the survival of the cells in the GCL. Sufficient number of functional ChR2 channels were maintained in ganglion cells to drive robust membrane depolarization and spike firing in response to light.
Expression of Chop2-GFP could be achieved in retinal neurons in vivo for the duration of the lifespan of mice. The expression of Chop2-GFP did not cause any detectable toxicity and cell death to neurons of the ganglion cell layer.
PMCID: PMC2730750  PMID: 19710944
10.  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.
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.
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.
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.
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
11.  Intraventricular Brain Injection of Adeno-Associated Virus Type 1 (AAV1) in Neonatal Mice Results in Complementary Patterns of Neuronal Transduction to AAV2 and Total Long-Term Correction of Storage Lesions in the Brains of β-Glucuronidase-Deficient Mice 
Journal of Virology  2003;77(12):7034-7040.
Inherited metabolic disorders that affect the central nervous system typically result in pathology throughout the brain; thus, gene therapy strategies need to achieve widespread delivery. We previously found that although intraventricular injection of the neonatal mouse brain with adeno-associated virus serotype 2 (AAV2) results in dispersed gene delivery, many brain structures were poorly transduced. This limitation may be overcome by using different AAV serotypes because the capsid proteins use different cellular receptors for entry, which may allow enhanced global targeting of the brain. We tested this with AAV1 and AAV5 vectors. AAV5 showed very limited brain transduction after neonatal injection, even though it has different transduction patterns than AAV2 in adult brain injections. In contrast, AAV1 vectors, which have not been tested in the brain, showed robust widespread transduction. Complementary patterns of transduction between AAV1 and AAV2 were established and maintained in the adult brain after neonatal injection. In the majority of structures, AAV1 transduced many more cells than AAV2. Both vectors transduced mostly neurons, indicating that differential expression of receptors on the surfaces of neurons occurs in the developing brain. The number of cells positive for a vector-encoded secreted enzyme (β-glucuronidase) was notably greater and more widespread in AAV1-injected brains. A comprehensive analysis of AAV1-treated brains from β-glucuronidase-deficient mice (mucopolysaccharidosis type VII) showed complete reversal of pathology in all areas of the brain for at least 1 year, demonstrating that the combination of this serotype and experimental strategy is therapeutically effective for treating global neurometabolic disorders.
PMCID: PMC156185  PMID: 12768022
12.  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.
PMCID: PMC2854182  PMID: 20138034
transgene expression; self-complementary AAV; scAAV5; AAV5; subretinal injection; intravitreal injection; photoreceptor; RPE
13.  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.
PMCID: PMC136354  PMID: 12097579
14.  Delivery of Adeno-Associated Virus Vectors to the Fetal Retina: Impact of Viral Capsid Proteins on Retinal Neuronal Progenitor Transduction 
Journal of Virology  2003;77(14):7957-7963.
The development of fetal ocular gene transfer may be useful as a therapeutic tool for the prevention of retinal genetic disorders with congenital or early clinical manifestations. In this study we explored the neural progenitor transduction patterns of adeno-associated virus (AAV) vectors following delivery to the developing retina. Recombinant vectors with the same genome carrying the enhanced green fluorescent protein (EGFP) transgene packaged in capsids of differing serotypes (serotypes 1, 2, and 5, termed AAV2/1, AAV2/2, and AAV2/5, respectively) were created. Delivery of the AAV vectors during early retinal development resulted in efficient and stable transduction of retinal progenitors. Vector surface proteins and the developmental status of the retina profoundly affected viral tropism and transgene distribution. The procedure is not detrimental to retinal development and function and therefore provides a safe delivery vehicle for potential therapeutic applications and a means of assessing the mechanisms of retina development and disease.
PMCID: PMC161923  PMID: 12829835
15.  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.
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.
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.
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.
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.
PMCID: PMC2939198  PMID: 20484599
16.  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.
PMCID: PMC3763646  PMID: 24281556
adeno-associated virus; retinal gene therapy; MFG-E8; αvβ5 integrin; photoreceptor outer segments
17.  Evaluation of AAV-Mediated Expression of Chop2-GFP in the Marmoset Retina 
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.
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.
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.
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.
PMCID: PMC2939198  PMID: 20484599
18.  Inverse zonation of hepatocyte transduction with AAV vectors between mice and non-human primates 
Molecular Genetics and Metabolism  2011;104(3):395-403.
Gene transfer vectors based on adeno-associated virus 8 (AAV8) are highly efficient in liver transduction and can be easily administered by intravenous injection. In mice, AAV8 transduces predominantly hepatocytes near central veins and yields lower transduction levels in hepatocytes in periportal regions. This transduction bias has important implications for gene therapy that aims to correct metabolic liver enzymes because metabolic zonation along the porto-central axis requires the expression of therapeutic proteins within the zone where they are normally localized.
In the present study we compared the expression pattern of AAV8 expressing green fluorescent protein (GFP) in liver between mice, dogs, and non-human primates. We confirmed the pericentral dominance in transgene expression in mice with AAV8 when the liver-specific thyroid hormone-binding globulin (TBG) promoter was used but also observed the same expression pattern with the ubiquitous chicken β-actin (CB) and cytomegalovirus (CMV) promoters, suggesting that transduction zonation is not caused by promoter specificity. Predominantly pericentral expression was also found in dogs injected with AAV8. In contrast, in cynomolgus and rhesus macaques the expression pattern from AAV vectors was reversed, i.e. transgene expression was most intense around portal areas and less intense or absent around central veins. Infant rhesus macaques as well as newborn mice injected with AAV8 however showed a random distribution of transgene expression with neither portal nor central transduction bias. Based on the data in monkeys, adult humans treated with AAV vectors are predicted to also express transgenes predominantly in periportal regions whereas infants are likely to show a uniform transduction pattern in liver.
PMCID: PMC3269907  PMID: 21778099
gene therapy; AAV; liver; animal models
19.  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.
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.
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.
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.
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
20.  Differential adeno-associated virus mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture 
Molecular Pain  2010;6:31.
Neuronal transduction by adeno-associated viral (AAV) vectors has been demonstrated in cortex, brainstem, cerebellum, and sensory ganglia. Intrathecal delivery of AAV serotypes that transduce neurons in dorsal root ganglia (DRG) and spinal cord offers substantial opportunities to 1) further study mechanisms underlying chronic pain, and 2) develop novel gene-based therapies for the treatment and management of chronic pain using a non-invasive delivery route with established safety margins. In this study we have compared expression patterns of AAV serotype 5 (AAV5)- and AAV serotype 8 (AAV8)-mediated gene transfer to sensory neurons following intrathecal delivery by direct lumbar puncture.
Intravenous mannitol pre-treatment significantly enhanced transduction of primary sensory neurons after direct lumbar puncture injection of AAV5 (rAAV5-GFP) or AAV8 (rAAV8-GFP) carrying the green fluorescent protein (GFP) gene. The presence of GFP in DRG neurons was consistent with the following evidence for primary afferent origin of the majority of GFP-positive fibers in spinal cord: 1) GFP-positive axons were evident in both dorsal roots and dorsal columns; and 2) dorsal rhizotomy, which severs the primary afferent input to spinal cord, abolished the majority of GFP labeling in dorsal horn. We found that both rAAV5-GFP and rAAV8-GFP appear to preferentially target large-diameter DRG neurons, while excluding the isolectin-B4 (IB4) -binding population of small diameter neurons. In addition, a larger proportion of CGRP-positive cells was transduced by rAAV5-GFP, compared to rAAV8-GFP.
The present study demonstrates the feasibility of minimally invasive gene transfer to sensory neurons using direct lumbar puncture and provides evidence for differential targeting of subtypes of DRG neurons by AAV vectors.
PMCID: PMC2900238  PMID: 20509925
21.  Anti-inflammatory effect of pigment epithelium-derived factor in DBA/2J mice 
Molecular Vision  2009;15:438-450.
Glaucoma is the second leading cause of blindness. The ultimate cause of vision loss in glaucoma is thought to be retinal ganglion cell (RGC) death. Neuroprotection of RGC is therefore an important goal of glaucoma therapy. Several lines of evidence suggest that pigment epithelium derived factor (PEDF) is a potent anti-angiogenic, neuroprotective, and anti-inflammatory factor for neurons. In this study, we examined the potential role of PEDF in protection of RGC in the DBA/2J mouse, an animal model of inherited glaucoma.
DBA/2J mice at two months of age were transfected intravitreally with adeno-associated virus (AAV)-PEDF or AAV-green fluorescent protein (AAV-GFP). RGC and nerve fiber layer protection were evaluated in retinal cross sections. Biochemical alterations in the retinas of DBA/2J mice in response to intravitreal transfection of PEDF were also examined by reverse transcriptase PCR (RT–PCR) and western blot. Cellular localization of PEDF and glial fibrillary acidic protein (GFAP) was determined by immunohistochemistry. Visual acuity was determined by optomotor testing.
PEDF protein levels in the retina and optic nerves of DBA/2J mice declined with age. The expression of tumor necrosis factor (TNF), GFAP, and interleukin-18 (IL-18) increased with age in the retina and optic nerve of DBA/2J mice. Intravitreal PEDF transfection in DBS/2J mice reduced loss of RGC and nerve fiber layer, delayed vision loss, and reduced TNF, IL-18, and GFAP expression in the retina and optic nerve.
Transduced PEDF potently and efficaciously reduces RGC loss and vision decline in DBA/2J mice, possibly via the reduction of TNF and IL-18, and downregulation of GFAP. The anti-inflammatory effect of PEDF represents a novel approach to the prevention of glaucomatous RGC death.
PMCID: PMC2647972  PMID: 19247457
22.  Administration of pigment epithelium-derived factor delivered by adeno-associated virus inhibits blood–retinal barrier breakdown in diabetic rats 
Molecular Vision  2010;16:2384-2394.
To evaluate the effect of the recombinant adeno-associated virus (rAAV) vector that expresses human pigment epithelium-derived factor (hPEDF) on reducing blood–retinal barrier (BRB) breakdown in the experimental diabetic rat model.
Diabetes was induced by an intraperitoneal (i.p.) injection of streptozotocin (STZ) into 10-week-old male Wister rats. rAAV2-cytomegalovirus (CMV)-hPEDF was delivered into the right eyes by intravitreal injection on the first day after diabetes induction. The contralateral eyes received intravitreal injection of rAAV2-CMV-green fluorescent protein as the paired control. Gene delivery and expression of vascular endothelial growth factor (VEGF), occludin, and intercellular adhesion molecule-1 (ICAM-1) were determined with reverse transciptase PCR or western blotting. BRB breakdown changes were quantified by measuring albumin leakage from retinal blood vessels after an intravenous (i.v.) injection of Evans blue albumin.
Retinal transfection with the hPEDF gene construct led to sustained hPEDF gene expression for 6 months, significantly suppressing VEGF mRNA expression in the retina after 1, 3, and 6 months of diabetes induced by STZ compared with paired controls. Moreover, hPEDF dramatically reduced the levels of retinal ICAM-1 but increased the expression of occludin. Furthermore, BRB breakdown was much lower in hPEDF-injected diabetic animals in comparison with controls after 6 months.
A single intravitreal injection of rAAV2-CMV-hPEDF can relieve BRB breakdown in STZ-induced diabetic rats for 6 months. The effect is associated with downregulation of retinal VEGF mRNA and ICAM-1 expression and a reduction in the loss of retinal occludin induced by diabetes. The approach of gene transfer may reduce diabetic macular edema, providing long-term protection for diabetic patients at risk of macular edema.
PMCID: PMC2994733  PMID: 21139695
23.  Adeno-associated virus serotype-9 efficiently transduces the retinal outer plexiform layer 
Molecular Vision  2009;15:1374-1382.
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.
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.
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.
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
24.  Expanded Repertoire of AAV Vector Serotypes Mediate Unique Patterns of Transduction in Mouse Brain 
A wide diversity of adeno-associated virus (AAV) structural proteins uncovered from latent genomes in primate tissue has expanded the number of AAV vector serotypes, which can potentially confer unique cell tropism to the vector. We evaluated 17 of these vectors in the mouse brain using green fluorescent protein (GFP) as a reporter gene. A rapid initial evaluation was performed by neonatal lateral ventricle injections. Vectors made with capsids hu.32, hu.37, pi.2, hu.11, rh.8, hu.48R3, and AAV9 for comparison were selected for further analysis based on their ability to transduce large numbers of cells and result in novel patterns of cell transduction. These vectors were injected into adult brains in four major structures (cortex, striatum, hippocampus, and thalamus), and all were found to transduce neurons. In addition, hu.32, hu.11, pi.2, hu.48R3, and rh.8 resulted in GFP expression in some astrocytes or oligodendrocytes. AAVs rh.8, pi.2, hu.32, and hu.11 also appeared to result in neuronal transport of the vector genome. Vector transport was studied by a single unilateral injection into the hippocampus and vector genome was found in projection sites of the hippocampus. These unique patterns of cell transduction expand the potential repertoire for targeting AAV vectors to selected subsets of brain cells.
PMCID: PMC3056207  PMID: 18714307
25.  Impact of Pre-Existing Immunity on Gene Transfer to Nonhuman Primate Liver with Adeno-Associated Virus 8 Vectors 
Human Gene Therapy  2011;22(11):1389-1401.
Vectors based on the primate-derived adeno-associated virus serotype 8 (AAV8) are being evaluated in preclinical and clinical models. Natural infections with related AAVs activate memory B cells that produce antibodies capable of modulating the efficacy and safety of the vector. We have evaluated the biology of AAV8 gene transfer in macaque liver, with a focus on assessing the impact of pre-existing humoral immunity. Twenty-one macaques with various levels of AAV neutralizing antibody (NAb) were injected intravenously with AAV8 vector expressing green fluorescent protein. Pre-existing antibody titers in excess of 1:10 substantially diminished hepatocyte transduction that, in the absence of NAbs, was highly efficient. Vector-specific NAb diminished liver deposition of genomes and unexpectedly increased genome distribution to the spleen. The majority of animals showed high-level and stable sequestration of vector capsid protein by follicular dendritic cells of splenic germinal centers. These studies illustrate how natural immunity to a virus that is related to a vector can impact the efficacy and potential safety of in vivo gene therapy. We propose to use the in vitro transduction inhibition assay to evaluate research subjects before gene therapy and to preclude from systemic AAV8 trials those that have titers in excess of 1:10.
Wang and colleagues evaluate the impact of preexisting humoral immunity on adeno-associated virus 8 (AAV8)-mediated gene transfer to macaque livers. They injected AAV8 vectors expressing green fluorescent protein into 21 macaques with various levels of AAV-neutralizing antibody (Nab), and found that NAb titers above 1:10 significantly impaired transduction and affected distribution of vector genomes.
PMCID: PMC3225046  PMID: 21476868

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