Tropisms of AAV for Subretinal Delivery to the Neonatal Mouse Retina and Its Application for In Vivo Rescue of Developmental Photoreceptor Disorders

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.     

A Single Intravenous AAV9 Injection Mediates Bilateral Gene Transfer to the Adult Mouse Retina

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.     

AAV-mediated gene therapy in mouse models of recessive retinal degeneration

In recent years, more and more mutant genes that cause retinal diseases have been detected. At the same time, many naturally occurring mouse models of retinal degeneration have also been found, which show similar changes to human retinal diseases. These, together with improved viral vector quality allow more and more traditionally incurable inherited retinal disorders to become potential candidates for gene therapy. Currently, the most common vehicle to deliver the therapeutic gene into target retinal cells is the adenoassociated viral vector (AAV). Following delivery to the immuno-privileged subretinal space, AAV-vectors can efficiently target both retinal pigment epithelium and photoreceptor cells, the origin of most retinal degenerations. This review focuses on the AAV-based gene therapy in mouse models of recessive retinal degenerations, especially those in which delivery of the correct copy of the wild-type gene has led to significant beneficial effects on visual function, as determined by morphological, biochemical, electroretinographic and behavioral analysis. The past studies in animal models and ongoing successful LCA2 clinical trials, predict a bright future for AAV gene replacement treatment for inherited recessive retinal diseases.     

Novel adeno-associated virus serotypes efficiently transduce murine photoreceptors

Severe inherited retinal diseases, such as retinitis pigmentosa and Leber congenital amaurosis, are caused by mutations in genes preferentially expressed in photoreceptors. While adeno-associated virus (AAV)-mediated gene transfer can correct retinal pigment epithelium (RPE) defects in animal models, approaches for the correction of photoreceptor-specific diseases are less efficient. We evaluated the ability of novel AAV serotypes (AAV2/7, AAV2/8, AAV2/9, AAV2rh.43, AAV2rh.64R1, and AAV2hu.29R) in combination with constitutive or photoreceptor-specific promoters to improve photoreceptor transduction, a limiting step in photoreceptor rescue. Based on a qualitative analysis, all AAV serotypes tested efficiently transduce the RPE as well as rod and cone photoreceptors after subretinal administration in mice. Interestingly, AAV2/9 efficiently transduces Müller cells. To compare photoreceptor transduction from different AAVs and promoters in both a qualitative and quantitative manner, we designed a strategy based on the use of a bicistronic construct expressing both enhanced green fluorescent protein and luciferase. We found that AAV2/8 and AAV2/7 mediate six- to eightfold higher levels of in vivo photoreceptor transduction than AAV2/5, considered so far the most efficient AAV serotype for photoreceptor targeting. In addition, following subretinal administration of AAV, the rhodopsin promoter allows significantly higher levels of photoreceptor expression than the other ubiquitous or photoreceptor-specific promoters tested. Finally, we show that AAV2/7, AAV2/8, and AAV2/9 outperform AAV2/5 following ex vivo transduction of retinal progenitor cells differentiated into photoreceptors. We conclude that AAV2/7 or AAV2/8 and the rhodopsin promoter provide the highest levels of photoreceptor transduction both in and ex vivo and that this may overcome the limitation to therapeutic success observed so far in models of inherited severe photoreceptor diseases.     

Virus-mediated transduction of murine retina with adeno-associated virus: effects of viral capsid and genome size

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.     

Comparative Analysis of DNA Nanoparticles and AAVs for Ocular Gene Delivery

Gene therapy is a critical tool for the treatment of monogenic retinal diseases. However, the limited vector capacity of the current benchmark delivery strategy, adeno-associated virus (AAV), makes development of larger capacity alternatives, such as compacted DNA nanoparticles (NPs), critical. Here we conduct a side-by-side comparison of self-complementary AAV and CK30PEG NPs using matched ITR plasmids. We report that although AAVs are more efficient per vector genome (vg) than NPs, NPs can drive gene expression on a comparable scale and longevity to AAV. We show that subretinally injected NPs do not leave the eye while some of the AAV-injected animals exhibited vector DNA and GFP expression in the visual pathways of the brain from PI-60 onward. As a result, these NPs have the potential to become a successful alternative for ocular gene therapy, especially for the multitude of genes too large for AAV vectors.     

Retinal Ganglion Cells are Resistant to Photoreceptor Loss in Retinal Degeneration

The rapid and massive degeneration of photoreceptors in retinal degeneration might have a dramatic negative effect on retinal circuits downstream of photoreceptors. However, the impact of photoreceptor loss on the morphology and function of retinal ganglion cells (RGCs) is not fully understood, precluding the rational design of therapeutic interventions that can reverse the progressive loss of retinal function. The present study investigated the morphological changes in several identified RGCs in the retinal degeneration rd1 mouse model of retinitis pigmentosa (RP), using a combination of viral transfection, microinjection of neurobiotin and confocal microscopy. Individual RGCs were visualized with a high degree of detail using an adeno-associated virus (AAV) vector carrying the gene for enhanced green fluorescent protein (EGFP), allowed for large-scale surveys of the morphology of RGCs over a wide age range. Interestingly, we found that the RGCs of nine different types we encountered were especially resistant to photoreceptor degeneration, and retained their fine dendritic geometry well beyond the complete death of photoreceptors. In addition, the RGC-specific markers revealed a remarkable degree of stability in both morphology and numbers of two identified types of RGCs for up to 18 months of age. Collectively, our data suggest that ganglion cells, the only output cells of the retina, are well preserved morphologically, indicating the ganglion cell population might be an attractive target for treating vision loss.     

Delivery of adeno-associated virus vectors to the fetal retina: impact of viral capsid proteins on retinal neuronal progenitor transduction

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.     

Adeno-associated virus gene expression inhibits cellular transformation by heterologous genes.

In this paper we report that adeno-associated virus (AAV) genomes inhibit stable transformation by several dominant selectable marker genes upon cotransfection into mouse tissue culture cells. Cotransfection of AAV genomes also inhibited the expression of pSV2cat in transient assays. In both cases, the inhibitory effect was independent of AAV DNA replication but required the AAV p5 and p19 genes, which encode proteins required for AAV DNA replication and regulation of AAV gene expression. Finally, addition of a cloned E4 gene in the transfection experiments partially blocked the AAV-mediated inhibitory activities.     

Novel AAV serotypes for improved ocular gene transfer

Some of the most successful gene therapy results have been obtained using recombinant viral vectors to treat animal models of inherited and acquired ocular diseases. Clinical trials using adenovirus vector systems have been initiated for two ocular diseases. Adeno-associated viruses (AAVs) represent an attractive alternative to adenoviral vector systems as they enable stable and long-term expression and can target a variety of different ocular cell types depending on the capsid serotype; recently clinical trails for congenital blindness was initiated with a vector-based AAV serotype 2. High levels of retinal gene transfer have been achieved using vectors based on AAV serotypes 1, 2, 4 and 5. This report compares the gene transfer efficacy and stability of expression of vector systems based on three novel AAV serotypes: AAV7, 8, 9, with the established vectors AAV1, 2, 5. We show here that AAV7 and 8 enable superior long-term transduction of retinal and also anterior chamber structures.     

Gene therapy and retinitis pigmentosa: advances and future challenges.

It may be possible, one day, to use gene therapy to treat diseases whose genetic defects have been discerned. Because many genes responsible for inherited eye disorders within the retina have been identified, diseases of the eye are prime candidates for this form of therapy. The eye also has the advantage of being highly accessible with altered immunological properties, important considerations for easy delivery of virus and avoidance of systemic immune responses. Currently, adenovirus, adeno-associated virus and lentivirus have been used to successfully transfer genetic material to retinal pigment epithelium and photoreceptor cells. By harnessing therapeutic genes to these viruses, researchers have been able to demonstrate rescue in rodent models of retinitis pigmentosa, providing evidence that this form of therapy can be effective in delaying photoreceptor cell death. Future challenges include confirming therapeutic effects in animal models with eyes more anatomically similar to those of humans and demonstrating long-term rescue with minimal toxicity.     

Impaired intracellular trafficking of adeno-associated virus type 2 vectors limits efficient transduction of murine fibroblasts

Although adeno-associated virus type 2 (AAV) has gained attention as a potentially useful alternative to the more commonly used retrovirus- and adenovirus-based vectors for human gene therapy, efficient gene transfer and transgene expression by AAV vectors require that the following two obstacles be overcome. First, the target cell must express the receptor and the coreceptor for AAV infection, and second, the cell must allow for viral second-strand DNA synthesis. We now describe a third obstacle, impaired intracellular trafficking of AAV to the nucleus, which results in the lack of transgene expression in murine fibroblasts which do express the AAV receptor and the coreceptor and which are permissive for viral second-strand DNA synthesis. We document that AAV vectors bind efficiently and gain entry successfully into NIH 3T3 cells, but trafficking into the nucleus is significantly impaired in these cells. In contrast, viral trafficking to the nucleus in cells known to be efficiently transduced by AAV vectors is both rapid and efficient. The demonstration of yet another obstacle in AAV-mediated gene transfer has implications for the optimal use of these vectors in human gene therapy.     

Factors Influencing Adeno-Associated Virus-Mediated Gene Transfer to Human Cystic Fibrosis Airway Epithelial Cells: Comparison with Adenovirus Vectors

Adeno-associated virus (AAV) vectors appear promising for use in gene therapy in cystic fibrosis (CF) patients, yet many features of AAV-mediated gene transfer to airway epithelial cells are not well understood. We compared the transduction efficiencies of AAV vectors and adenovirus (Ad) vectors in immortalized cell lines from CF patients and in nasal epithelial primary cultures from normal humans and CF patients. Similar dose-dependent relationships between the vector multiplicities of infection and the efficiencies of lacZ gene transfer were observed. However, levels of transduction for both Ad and recombinant AAV (rAAV) were significantly lower in the airway epithelial cell than in the control cell lines HeLa and HEK 293. Transduction efficiencies differed among cultured epithelial cell types, with poorly differentiated cells transducing more efficiently than well-differentiated cells. A time-dependent increase in gene expression was observed after infection for both vectors. For Ad, but not for AAV, this increase was dependent on prolonged incubation of cells with the vector. Furthermore, for rAAV (but not for rAd), the delay in maximal transduction could be abrogated by wild-type Ad helper infection. Thus, although helper virus is not required for maximal transduction, it increases the kinetics by which this is achieved. Expression of Ad E4 open reading frame 6 or addition of either hydroxyurea or camptothecin resulted in increased AAV transduction, as previously demonstrated for nonairway cells (albeit to lower final levels), suggesting that second-strand synthesis may not be the sole cause of inefficient transduction. Finally, the efficiency of AAV-mediated ex vivo gene transfer to lung cells was similar to that previously described for Ad vectors in that transduction was limited to regions of epithelial injury and preferentially targeted basal-like cells. These studies address the primary factors influencing rAAV infection of human airway cells and should impact successful gene delivery in CF patients.     

Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma

Glaucoma is one of the leading eye diseases resulting in blindness due to the death of retinal ganglion cells. This study aimed to develop novel protocol to promote the differentiation of retinal Müller cells into ganglion cells in vivo in a rat model of glaucoma. The stem cells dedifferentiated from rat retinal Müller cells were randomized to receive transfection with empty lentivirus PGC-FU-GFP or lentivirus PGC-FU-Atoh7-GFP, or no transfection. The stem cells were induced further to differentiate. Ocular hypertension was induced using laser photocoagulation. The eyes were injected with Atoh7 expression vector lentivirus PGC-FU-Atoh7-GFP. Eyeball frozen sections, immunohistochemistry, RT-PCR, Western bolt, and apoptosis assay were performed. We found that the proportion of ganglion cells differentiated from Atoh7-tranfected stem cells was significantly higher than that of the other two groups. The mean intraocular pressure of glaucomatous eyes was elevated significantly compared with those of contralateral eyes. Some retinal Müller cells in the inner nuclear layer entered the mitotic cell cycle in rat chronic ocular hypertension glaucoma model. Atoh7 contributes to the differentiation of retinal Müller cells into retinal ganglion cells in rat model of glaucoma. In conclusion, Atoh7 promotes the differentiation of Müller cells-derived retinal stem cells into retinal ganglion cells in a rat model of glaucoma, thus opening up a new avenue for gene therapy and optic nerve regeneration in glaucoma.     

CD8(+) T-cell responses to adeno-associated virus capsid in humans

Hepatic adeno-associated virus (AAV)-serotype 2 mediated gene transfer results in transgene product expression that is sustained in experimental animals but not in human subjects. We hypothesize that this is caused by rejection of transduced hepatocytes by AAV capsid-specific memory CD8(+) T cells reactivated by AAV vectors. Here we show that healthy subjects carry AAV capsid-specific CD8(+) T cells and that AAV-mediated gene transfer results in their expansion. No such expansion occurs in mice after AAV-mediated gene transfer. In addition, we show that AAV-2 induced human T cells proliferate upon exposure to alternate AAV serotypes, indicating that other serotypes are unlikely to evade capsid-specific immune responses.     

Human gene targeting by adeno-associated virus vectors is enhanced by DNA double-strand breaks

The use of adeno-associated virus (AAV) to package gene-targeting vectors as single-stranded linear molecules has led to significant improvements in mammalian gene-targeting frequencies. However, the molecular basis for the high targeting frequencies obtained is poorly understood, and there could be important mechanistic differences between AAV-mediated gene targeting and conventional gene targeting with transfected double-stranded DNA constructs. Conventional gene targeting is thought to occur by the double-strand break (DSB) model of homologous recombination, as this can explain the higher targeting frequencies observed when DSBs are present in the targeting construct or target locus. Here we compare AAV-mediated gene-targeting frequencies in the presence and absence of induced target site DSBs. Retroviral vectors were used to introduce a mutant lacZ gene containing an I-SceI cleavage site and to efficiently deliver the I-SceI endonuclease, allowing us to carry out these studies with normal and transformed human cells. Creation of DSBs by I-SceI increased AAV-mediated gene-targeting frequencies 60- to 100-fold and resulted in a precise correction of the mutant lacZ reporter gene. These experiments demonstrate that AAV-mediated gene targeting can result in repair of a DNA DSB and that this form of gene targeting exhibits fundamental similarities to conventional gene targeting. In addition, our findings suggest that the selective creation of DSBs by using viral delivery systems can increase gene-targeting frequencies in scientific and therapeutic applications.     

Infectious entry pathway of adeno-associated virus and adeno-associated virus vectors

We have investigated the infectious entry pathway of adeno-associated virus (AAV) and recombinant AAV vectors by assessing AAV-mediated gene transfer and by covalently conjugating fluorophores to AAV and monitoring entry by fluorescence microscopy. We examined AAV entry in HeLa cells and in HeLa cell lines which inducibly expressed a dominant interfering mutant of dynamin. The data demonstrate that AAV internalizes rapidly by standard receptor-mediated endocytosis from clathrin-coated pits (half-time <10 min). The lysosomotropic agents ammonium chloride and bafilomycin A(1) prevent AAV-mediated gene transfer when present during the first 30 min after the onset of endocytosis, indicating that AAV escapes from early endosomes yet requires an acidic environment for penetration into the cytosol. Following release from the endosome, AAV rapidly moves to the cell nucleus and accumulates perinuclearly beginning within 30 min after the onset of endocytosis. We present data indicating that escape of AAV from the endosome and trafficking of viral particles to the nucleus are unaffected by the presence of adenovirus, the primary helper virus for a productive AAV infection. Within 2 h, viral particles could be detected within the cell nucleus, suggesting that AAV enters the nucleus prior to uncoating. Interestingly, the majority of the intracellular virus particles remain in a stable perinuclear compartment even though gene expression from nuclear AAV genomes can be detected. This suggests that the process of nuclear entry is rate limiting or that AAV entry involves multiple pathways. Nevertheless, these data establish specific points in the AAV infectious entry process and have allowed the generation of a model for future expansion to specific cell types and AAV vector analysis in vivo.     

High-Fidelity Correction of Mutations at Multiple Chromosomal Positions by Adeno-Associated Virus Vectors

The gene targeting techniques used to modify chromosomes in mouse embryonic stem cells have had limited success with many other cell types, especially normal primary cells with restricted growth capacity outside the organism. This is due in large part to the technical problems and/or inefficiency of conventional DNA transfer methods, as well as the low rates of homologous recombination obtained in unselected cell populations. We recently described an alternative approach in which adeno-associated virus (AAV) vectors were used to modify homologous chromosomal sequences, and targeting rates close to 1% were observed at the single copy hypoxanthine phosphoribosyl transferase (HPRT) locus in normal human cells (D. W. Russell and R. K. Hirata, Nat. Genet. 18:325-330, 1998). Here we report experiments in which we used a retroviral shuttle vector system to introduce and characterize target loci in human chromosomes, and demonstrate that AAV vectors can correct several types of mutations with high fidelity, independent of chromosomal position. The gene targeting rates varied depending on the type of mutation being corrected, implicating cellular mismatch recognition functions in the reaction. Since AAV vectors can efficiently deliver DNA to many cell types both in vivo and ex vivo, our results suggest that AAV-mediated gene targeting will have wide applicability, including therapeutic gene correction.     

Efficient and persistent transduction of exocrine and endocrine pancreas by adeno-associated virus type 8

Efficient delivery of therapeutic proteins into the pancreas represents a major obstacle to gene therapy of pancreatic disorders. The current study compared the efficiency of recombinant lentivirus and adeno-associated virus (AAV) serotypes 1, 2, 5, 8 vectors delivered by intrapancreatic injection for gene transfer in vivo. Our results indicate that lentivirus and AAV 1, 2, 8 are capable of transducing pancreas with the order of efficiency AAV8 >>AAV1 > AAV2 ≥ lentivirus, whereas AAV5 was ineffective. AAV8 resulted in an efficient, persistent (150 days) and dose-dependent transduction in exocrine acinar cells and endocrine islet cells. Pancreatic ducts and blood vessels were also transduced. Extrapancreatic transduction was restricted to liver. Leukocyte infiltration was not observed in pancreas and blood glucose levels were not altered. Thus, AAV8 represents a safe and effective vehicle for therapeutic gene transfer to pancreas in vivo.