scAAV9 intracisternal delivery results in efficient gene transfer to the central nervous system of a feline model of motor neuron disease.

On the basis of previous studies suggesting that vascular endothelial growth factor (VEGF) could protect motor neurons from degeneration, adeno-associated virus vectors (serotypes 1 and 9) encoding VEGF (AAV.vegf) were administered in a limb-expression 1 (LIX1)-deficient cat-a large animal model of lower motor neuron disease-using three different delivery routes to the central nervous system. AAV.vegf vectors were injected into the motor cortex via intracerebral administration, into the cisterna magna, or intravenously in young adult cats. Intracerebral injections resulted in detectable transgene DNA and transcripts throughout the spinal cord, confirming anterograde transport of AAV via the corticospinal pathway. However, such strategy led to low levels of VEGF expression in the spinal cord. Similar AAV doses injected intravenously resulted also in poor spinal cord transduction. In contrast, intracisternal delivery of AAV exhibited long-term transduction and high levels of VEGF expression in the entire spinal cord, yet with no detectable therapeutic clinical benefit in LIX1-deficient animals. Altogether, we demonstrate (i) that intracisternal delivery is an effective AAV delivery route resulting in high transduction of the entire spinal cord, associated with little to no off-target gene expression, and (ii) that in a LIX1-deficient cat model, however, VEGF expressed at high levels in the spinal cord has no beneficial impact on the disease course.

Neonatal systemic delivery of scAAV9 in rodents and large animals results in gene transfer to RPE cells in the retina.

Systemic delivery of recombinant adeno-associated virus (rAAV) vectors has recently been shown to cross the blood brain barrier in rodents and large animals and to efficiently target cells of the central nervous system. Such approach could be particularly interesting to treat lysosomal storage diseases or neurodegenerative disorders characterized by multiple organs injuries especially neuronal and retinal dysfunctions. However, the ability of rAAV vector to cross the blood retina barrier and to transduce retinal cells after systemic injection has not been precisely determined. In this study, gene transfer was investigated in the retina of neonatal and adult rats after intravenous injection of self-complementary (sc) rAAV serotype 1, 5, 6, 8, and 9 carrying a CMV-driven green fluorescent protein (GFP), by fluorescence fundus photography and histological examination. Neonatal rats injected with scAAV2/9 vector displayed the strongest GFP expression in the retina, within the retinal pigment epithelium (RPE) cells. Retinal tropism of scAAV2/9 vector was further assessed after systemic delivery in large animal models, i.e., dogs and cats. Interestingly, efficient gene transfer was observed in the RPE cells of these two large animal models following neonatal intravenous injection of the vector. The ability of scAAV2/9 to transduce simultaneously neurons in the central nervous system, and RPE cells in the retina, after neonatal systemic delivery, makes this approach potentially interesting for the treatment of infantile neurodegenerative diseases characterized by both neuronal and retinal damages.

Subretinal delivery of recombinant AAV serotype 8 vector in dogs results in gene transfer to neurons in the brain.

Recombinant adeno-associated virus (rAAV) vectors are among the most efficient gene delivery vehicles for gene transfer to the retina. This study evaluates the behavior of the rAAV8 serotype vector with regard to intraocular delivery in rats and dogs. Subretinal delivery of an AAV2/8.gfp vector results in efficient gene transfer in the retinal pigment epithelium (RPE), the photoreceptors and, surprisingly, in the cells of the inner nuclear layer as well as in ganglion cells. Most importantly, in dogs, gene transfer also occurred distal to the injection site in neurons of the lateral geniculate nucleus of the brain. Because green fluorescent protein (GFP) was detected along the visual pathway within the brain, we analyzed total DNA extracted from various brain slices using PCR. Vector sequences were detected in many parts of the brain, but chiefly in the contralateral hemisphere.

Long-term doxycycline-regulated transgene expression in the retina of nonhuman primates following subretinal injection of recombinant AAV vectors.

Adeno-associated viral gene therapy has shown promise for the treatment of inherited and acquired retinal disorders. In most applications, regulation of expression is a critical concern for both safety and efficacy. The purpose of our study was to evaluate the ability of the tetracycline-regulatable system to establish long-term transgene regulation in the retina of nonhuman primates. Three rAAV vectors expressing the tetracycline-dependent transactivator (rtTA) under the control of either the ubiquitous CAG promoter or the specific RPE65 promoter (AAV2/5.CAG.TetOn.epo, AAV2/4.CAG.TetOn.epo, and AAV2/4.RPE65.TetOn.epo) were generated and administered subretinally to seven macaques. We demonstrated that repeated inductions of transgene expression in the nonhuman primate retina can be achieved using a Tet-inducible system via rAAV vector administration over a long period (2.5 years). Maximum erythropoietin (EPO) secretion in the anterior chamber depends upon the rAAV serotype and the nature of the promoter driving rtTA expression. We observed that the EPO isoforms produced in the retina differ from one another based on the transduced cell type of origin within the retina and also differ from both the physiological EPO isoforms and the isoforms produced by AAV-transduced skeletal muscle.

Gene therapeutic prospects in early onset of severe retinal dystrophy: restoration of vision in RPE65 Briard dogs using an AAV serotype 4 vector that specifically targets the retinal pigmented epithelium.

Previous studies have tested gene replacement therapy in RPE65 deficient dogs using recombinant adeno-associated virus 2/2 (rAAV2/2), -2/1 or -2/5 mediated delivery of the RPE65 gene. They all documented restoration of dark- and light-adapted ERG responses and improved psychophysical outcomes. Use of a specific RPE65 promoter and a rAAV vector that targets transgene expression specifically to the retinal pigmented epithelium (RPE) may, however, provide a safer setting for the long-term therapeutic expression of RPE65. Subretinal injection of rAAV2 pseudotyped with serotype 4 (rAAV2/4) specifically targets the RPE. The purpose of our study was to evaluate a rAAV2/4 vector carrying a human RPE65cDNA driven by a human RPE65 promoter, for the ability to restore vision in RPE65-/- purebred Briard dogs. Recombinant rAAV2/4 and rAAV2/2 vectors containing similar human RPE65 promoter and cDNA cassettes were generated and administered subretinally in 8 affected dogs, ages 8 to 30 months (n = 6 with rAAV2/4, n = 2 with rAAV2/2). Although fluorescein angiography and OCT examinations displayed retinal abnormalities in treated retinas, electrophysiological analysis demonstrated that restoration of rod and cone photoreceptor function started as soon as 15 days post-injection, reaching maximal function at 3 months post-injection, and remaining stable thereafter in all animals treated at 8 to 11 months of age. As assessed by the ability of these animals to avoid obstacles in both dim and normal light, functional vision was restored in the treated eye, while the untreated contralateral eye served as an internal control. The dog treated at a later age (30 months) did not recover retinal function or vision, suggesting that there might be a therapeutic window for the successful treatment of RPE65 -/- dogs by gene replacement therapy.

Postsurgical assessment and long-term safety of recombinant adeno-associated virus-mediated gene transfer into the retinas of dogs and primates.

OBJECTIVE: To evaluate, in dogs and primates, the short-term effects of subretinal injection and the safety of long-term recombinant adeno-associated virus (rAAV)-mediated transgene expression with respect to retinal morphology and function. METHODS: Subretinal delivery of rAAV (serotype 2, 4, or 5) was performed unilaterally in 14 beagles and 9 macaques. Postsurgical condition was evaluated during a 2-month follow-up study. Three dogs and 1 primate were examined for the long-term study. Green fluorescent protein expression was monitored by fluorescent retinal imaging. Retinal anatomy and function were assessed by angiography and electroretinography, respectively. RESULTS: Transgene expression was observed in 20 of 23 subretinally injected animals (both with and without vitrectomy). We did not detect an inflammatory response in any of the 23 treated subjects. In the long-term study, transgene expression was detected at the latest points evaluated: 36 months for the rAAV-2-injected dog, 24 months for the rAAV-4 and rAAV-5 dogs, and more than 18 months for the rAAV-4-injected primate. Angiography examinations were performed and showed no retinal abnormalities. Functional evaluation showed normal electroretinographic amplitude responses that were similar to those of the noninjected contralateral eyes. CONCLUSIONS: Subretinal injection of the rAAV vector in dogs and primates is a safe procedure with no perioperative complications and a high rate of successful retinal gene transfer. The retinal anatomy and function remained unchanged, despite persistent transgene expression up to 36 months postinjection with rAAV-2, -4, or -5. Additionally, we observed no other adverse effects, such as tumor formation due to possible insertional mutagenesis. These short- and long-term studies on rAAV transgene expression using large animals are encouraging for the prospects of ocular gene therapy applications in humans. CLINICAL RELEVANCE: These short- and long-term studies on rAAV transgene expression using large animals are encouraging for the prospects of ocular gene therapy applications in humans.