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Gene delivery vectors based on adeno-associated viruses (AAV) have exhibited promise in both preclinical disease models and human clinical trials for numerous disease targets, including the retinal degenerative disorders Leber’s congenital amaurosis and choroideremia. One general challenge for AAV is that preexisting immunity, as well as subsequent development of immunity following vector administration, can severely inhibit systemic AAV vector gene delivery. However, the role of neutralizing antibodies (NABs) in AAV transduction of tissues considered to be immune privileged, such as the eye, is unclear in large animals. Intravitreal AAV administration allows for broad retinal delivery, but is more susceptible to interactions with the immune system than subretinal administration. To assess the effects of systemic anti-AAV antibody levels on intravitreal gene delivery, we quantified the anti-AAV antibodies present in sera from non-human primates before and after intravitreal injections with various AAV capsids. Analysis showed that intravitreal administration resulted in an increase in anti-AAV antibodies regardless of the capsid serotype, transgene or dosage of virus injected. For monkeys injected with wild-type AAV2 and/or an AAV2 mutant, the variable that most significantly affected the production of anti-AAV2 antibodies was the amount of virus delivered. In addition, post-injection antibody titers were highest against the serotype administered, but the antibodies were also cross-reactive against other AAV serotypes. Furthermore, NAB levels in serum correlated with those in vitreal fluid, demonstrating both that this route of administration exposes AAV capsid epitopes to the adaptive immune system and that serum measurements are predictive of vitreous fluid NAB titers. Moreover, the presence of preexisting NAB titers in the serum of monkeys correlated strongly ( R =0.76) with weak, decaying or no transgene expression following intravitreal administration of AAV. Investigating anti-AAV antibody development will aid in understanding the interactions between gene therapy vectors and the immune system during ocular administration and can form a basis for future clinical studies applying intravitreal gene delivery.

Delivery of therapeutic genes to a large region of the retina with minimal damage from intraocular surgery is a central goal of treatment for retinal degenerations. Recent studies have shown that AAV9 can reach the central nervous system (CNS) and retina when administered systemically to neonates, which is a promising strategy for some retinal diseases. We investigated whether the retinal transduction efficiency of systemically delivered AAV9 could be improved by mutating capsid surface tyrosines, previously shown to increase the infectivity of several AAV vectors. Specifically, we evaluated retinal transduction following neonatal intravascular administration of AAV9 vectors containing tyrosine to phenylalanine mutations at two highly conserved sites. Our results show that a novel, double tyrosine mutant of AAV9 significantly enhanced gene delivery to the CNS and retina, and that gene expression can be restricted to rod photoreceptor cells by incorporating a rhodopsin promoter. This approach provides a new methodology for the development of retinal gene therapies or creation of animal models of neurodegenerative disease.

X-linked retinoschisis, a disease characterized by splitting of the retina, is caused by mutations in the retinoschisin gene, which encodes a putative secreted cell adhesion protein. Currently, there is no effective treatment for retinoschisis, though viral vector-mediated gene replacement therapies offer promise. We used intravitreal delivery of three different AAV vectors to target delivery of the RS1 gene to Müller glia, photoreceptors or multiple cell types throughout the retina. Müller glia radially span the entire retina, are accessible from the vitreous, and remain intact throughout progression of the disease. However, photoreceptors, not glia, normally secrete retinoschisin. We compared the efficacy of rescue mediated by retinoschisin secretion from these specific subtypes of retinal cells in the Rs1h −/− mouse model of retinoschisis. Our results indicate that all three vectors deliver the RS1 gene, and that several cell types can secrete retinoschisin, leading to transport of the protein across the retina. The greatest long-term rescue was observed when photoreceptors produce retinoschisin. Similar rescue was observed with photoreceptor-specific or generalized expression, although photoreceptor secretion may contribute to rescue in the latter case. These results collectively point to the importance of cell targeting and appropriate vector choice in the success of retinal gene therapies.

The use of the visual system played a major role in the elucidation of molecular mechanisms controlling axonal regeneration in the injured CNS after trauma. In this model, CNTF was shown to be the most potent known neurotrophic factor for axonal regeneration in the injured optic nerve. To clarify the role of the downstream growth regulator Stat3, we analyzed axonal regeneration and neuronal survival after an optic nerve crush in adult mice. The infection of retinal ganglion cells with adeno-associated virus serotype 2 (AAV2) containing wild-type (Stat3-wt) or constitutively active (Stat3-ca) Stat3 cDNA promoted axonal regeneration in the injured optic nerve. Axonal growth was analyzed in whole-mounted optic nerves in three dimensions (3D) after tissue clearing. Surprisingly, with AAV2.Stat3-ca stimulation, axons elongating beyond the lesion site displayed very irregular courses, including frequent U-turns, suggesting massive directionality and guidance problems. The pharmacological blockade of ROCK, a key signaling component for myelin-associated growth inhibitors, reduced axonal U-turns and potentiated AAV2.Stat3-ca-induced regeneration. Similar results were obtained after the sustained delivery of CNTF in the axotomized retina. These results show the important role of Stat3 in the activation of the neuronal growth program for regeneration, and they reveal that axonal misguidance is a key limiting factor that can affect long-distance regeneration and target interaction after trauma in the CNS. The correction of axonal misguidance was associated with improved long-distance axon regeneration in the injured adult CNS.

We describe a general approach for achieving efficient and cell type-specific expression of exogenous genes in photoreceptor cells of the mammalian retina. Recombinant adeno-associated virus (rAAV) vectors were used to transfer the bacterial lacZ gene or a synthetic green fluorescent protein gene (gfp) to mouse or rat retinas after injection into the subretinal space. Using a proximal murine rod opsin promoter (+86 to -385) to drive expression, reporter gene product was found exclusively in photoreceptors, not in any other retinal cell type or in the adjacent retinal pigment epithelium. GFP-expressing photoreceptors typically encompassed 10-20% of the total retinal area after a single 2-μ l injection. Photoreceptors were transduced with nearly 100% efficiency in the region directly surrounding the injection site. We estimate approximately 2.5 million photoreceptors were transduced as a result of the single subretinal inoculation. This level of gene transfer and expression suggests the feasibility of genetic therapy for retinal disease. The gfp-containing rAAV stock was substantially free of both adenovirus and wild-type AAV, as judged by plaque assay and infectious center assay, respectively. Thus, highly purified, helper virus-free rAAV vectors can achieve high-frequency tissue-specific transduction of terminally differentiated, postmitotic photoreceptor cells.

Nogo-A, an axonal growth inhibitory protein known to be mostly present in CNS myelin, was upregulated in retinal ganglion cells (RGCs) after optic nerve injury in adult mice. Nogo-A increased concomitantly with the endoplasmic reticulum stress (ER stress) marker C/EBP homologous protein (CHOP), but CHOP immunostaining and the apoptosis marker annexin V did not co-localize with Nogo-A in individual RGC cell bodies, suggesting that injury-induced Nogo-A upregulation is not involved in axotomy-induced cell death. Silencing Nogo-A with an adeno-associated virus serotype 2 containing a short hairpin RNA (AAV2.shRNA-Nogo-A) or Nogo-A gene ablation in knock-out (KO) animals had little effect on the lesion-induced cell stress or death. On the other hand, Nogo-A overexpression mediated by AAV2.Nogo-A exacerbated RGC cell death after injury. Strikingly, however, injury-induced sprouting of the cut axons and the expression of growth-associated molecules were markedly reduced by AAV2.shRNA-Nogo-A. The axonal growth in the optic nerve activated by the intraocular injection of the inflammatory molecule Pam3Cys tended to be lower in Nogo-A KO mice than in WT mice. Nogo-A overexpression in RGCs in vivo or in the neuronal cell line F11 in vitro promoted regeneration, demonstrating a positive, cell-autonomous role for neuronal Nogo-A in the modulation of axonal regeneration.

Viral delivery of neurotrophins or other therapeutic genes is an attractive option for treating retinal degeneration. Regulated expression of these genes in the retina is needed to aid in dose delivery and to promote safety. To evaluate whether tetracycline (tet)-inducible transgenes encapsidated in recombinant adeno-associated viruses (rAAV) can provide controlled gene expression in vitro and in the rat retina, two viruses were constructed: a silencer/activator vector and an inducible doxycycline (dox)-responsive GFP vector. Combinations of these two viruses were subretinally injected into wild-type rats and dox was orally administered through the drinking water. Retinal GFP expression was monitored in vivo with a noninvasive fluorescence imaging method. Eyes were also examined by histology, Western analysis, and electroretinography. Subretinal injection of rAAV efficiently delivers inducible genes to both photoreceptors and retinal pigment epithelial cells. GFP expression was initially observed 1 week postinduction, and GFP protein was undetectable after removal of dox. In uninduced animals, GFP expression was negligible. The dox dosage was varied in vivo and showed a correlation to the level of GFP expression. Thus, transduction of retinal cells with tet-inducible vectors allows for tight regulation of gene expression.

Apoptosis of photoreceptors occurs infrequently in adult retina but can be triggered in inherited and environmentally induced retinal degenerations. The protooncogene bcl-2 is known to be a potent regulator of cell survival in neurons. We created lines of transgenic mice overexpressing bcl-2 to test for its ability to increase photoreceptor survival. Bcl-2 increased photoreceptor survival in mice with retinal degeneration caused by a defective opsin or cGMP phosphodiesterase. Overexpression of Bcl-2 in normal photoreceptors also decreased the damaging effects of constant light exposure. Apoptosis was induced in normal photoreceptors by very high levels of bcl-2. We conclude that bcl-2 is an important regulator of photoreceptor cell death in retinal degenerations.

There is much debate on the adeno-associated virus (AAV) serotype that best targets specific retinal cell types and the route of surgical delivery—intravitreal or subretinal. This study compared three of the most efficacious AAV vectors known to date in a mouse model of retinal degeneration ( rd1 mouse) and macaque and human retinal explants. Green fluorescent protein (GFP) driven by a ubiquitous promoter was packaged into three AAV capsids: AAV2/8(Y733F), AAV2/2(quad Y-F) and AAV2/2(7m8). Overall, AAV2/2(7m8) transduced the largest area of retina and resulted in the highest level of GFP expression, followed by AAV2/2(quad Y-F) and AAV2/8(Y733F). AAV2/2(7m8) and AAV2/2(quad Y-F) both resulted in similar patterns of transduction whether they were injected intravitreally or subretinally. AAV2/8(Y733F) transduced a significantly smaller area of retina when injected intravitreally compared with subretinally. Retinal ganglion cells, horizontal cells and retinal pigment epithelium expressed relatively high levels of GFP in the mouse retina, whereas amacrine cells expressed low levels of GFP and bipolar cells were infrequently transduced. Cone cells were the most frequently transduced cell type in macaque retina explants, whereas Müller cells were the predominant transduced cell type in human retinal explants. Of the AAV serotypes tested, AAV2/2(7m8) was the most effective at transducing a range of cell types in degenerate mouse retina and macaque and human retinal explants.

We designed experiments to evaluate the therapeutic potential of glial cell line derived neurotrophic factor (GDNF) to rescue photoreceptors from genetically determined cell death. Gene transfer of the neurotrophic factor to the retina was achieved via a recombinant adeno-associated virus (rAAV) vector containing the chicken β-actin promoter/immediate early cytomegalovirus enhancer (CBA) driving the human GDNF gene. We delivered AAV-CBA-GDNF to the retinas of an animal model of retinitis pigmentosa, the TgN S334ter-4 rhodopsin line of transgenic rats. Immunohistochemical studies localized AAV-CBA-GDNF-derived recombinant protein to cell bodies, inner segments, and outer segments of photoreceptor cells as well as to retinal pigment epithelial cells. We assessed the effect of viral delivery by morphometric and electroretinographic analysis. These experiments showed that GDNF vector treatment leads to increased rod photoreceptor survival as indicated by morphometric analysis of outer nuclear layer thickness. AAV-CBA-GDNF-treated retinas also demonstrated functional improvement by the substantially increased amplitude of electroretinograms. AAV-CBA-GDNF delivery had a significant rescue effect on photoreceptor degeneration in this animal model.