Gene Therapy Regenerates Protein Expression in Cone Photoreceptors in Rpe65R91W/R91W Mice

Cone photoreceptors mediate visual acuity under daylight conditions, so loss of cone-mediated central vision of course dramatically affects the quality of life of patients suffering from retinal degeneration. Therefore, promoting cone survival has become the goal of many ocular therapies and defining the stage of degeneration that still allows cell rescue is of prime importance. Using the Rpe65R91W/R91W mouse, which carries a mutation in the Rpe65 gene leading to progressive photoreceptor degeneration in both patients and mice, we defined stages of retinal degeneration that still allow cone rescue. We evaluated the therapeutic window within which cones can be rescued, using a subretinal injection of a lentiviral vector driving expression of RPE65 in the Rpe65R91W/R91W mice. Surprisingly, when applied to adult mice (1 month) this treatment not only stalls or slows cone degeneration but, actually, induces cone-specific protein expression that was previously absent. Before the intervention only part of the cones (40% of the number found in wild-type animals) in the Rpe65R91W/R91W mice expressed cone transducin (GNAT2); this fraction increased to 64% after treatment. Correct S-opsin localization is also recovered in the transduced region. In consequence these results represent an extended therapeutic window compared to the Rpe65-/- mice, implying that patients suffering from missense mutations might also benefit from a prolonged therapeutic window. Moreover, cones are not only rescued during the course of the degeneration, but can actually recover their initial status, meaning that a proportion of altered cones in chromophore deficiency-related disease can be rehabilitated even though they are severely affected

Apolipoprotein E promotes subretinal mononuclear phagocyte survival and chronic inflammation in age-related macular degeneration

International audiencePhysiologically, the retinal pigment epithelium (RPE) expresses immunosuppressive signals such as FAS ligand (FASL), which prevents the accumulation of leukocytes in the subretinal space. Age-related macular degeneration (AMD) is associated with a breakdown of the subretinal immunosuppressive environment and chronic accumulation of mononuclear phagocytes (MPs). We show that subretinal MPs in AMD patients accumulate on the RPE and express high levels of APOE. MPs of Cx3cr1 À/À mice that develop MP accumulation on the RPE, photoreceptor degeneration, and increased choroidal neovascularization similarly express high levels of APOE. ApoE deletion in Cx3cr1 À/À mice prevents patho-genic age-and stress-induced subretinal MP accumulation. We demonstrate that increased APOE levels induce IL-6 in MPs via the activation of the TLR2-CD14-dependent innate immunity receptor cluster. IL-6 in turn represses RPE FasL expression and prolongs subretinal MP survival. This mechanism may account, in part, for the MP accumulation observed in Cx3cr1 À/À mice. Our results underline the inflammatory role of APOE in sterile inflammation in the immunosuppressive subretinal space. They provide rationale for the implication of IL-6 in AMD and open avenues toward therapies inhibiting pathogenic chronic inflammation in late AMD

Lentiviral gene transfer of RPE65 rescues survival and function of cones in a mouse model of Leber congenital amaurosis.

BACKGROUND: RPE65 is specifically expressed in the retinal pigment epithelium and is essential for the recycling of 11-cis-retinal, the chromophore of rod and cone opsins. In humans, mutations in RPE65 lead to Leber congenital amaurosis or early-onset retinal dystrophy, a severe form of retinitis pigmentosa. The proof of feasibility of gene therapy for RPE65 deficiency has already been established in a dog model of Leber congenital amaurosis, but rescue of the cone function, although crucial for human high-acuity vision, has never been strictly proven. In Rpe65 knockout mice, photoreceptors show a drastically reduced light sensitivity and are subject to degeneration, the cone photoreceptors being lost at early stages of the disease. In the present study, we address the question of whether application of a lentiviral vector expressing the Rpe65 mouse cDNA prevents cone degeneration and restores cone function in Rpe65 knockout mice. METHODS AND FINDINGS: Subretinal injection of the vector in Rpe65-deficient mice led to sustained expression of Rpe65 in the retinal pigment epithelium. Electroretinogram recordings showed that Rpe65 gene transfer restored retinal function to a near-normal pattern. We performed histological analyses using cone-specific markers and demonstrated that Rpe65 gene transfer completely prevented cone degeneration until at least four months, an age at which almost all cones have degenerated in the untreated Rpe65-deficient mouse. We established an algorithm that allows prediction of the cone-rescue area as a function of transgene expression, which should be a useful tool for future clinical trials. Finally, in mice deficient for both RPE65 and rod transducin, Rpe65 gene transfer restored cone function when applied at an early stage of the disease. CONCLUSIONS: By demonstrating that lentivirus-mediated Rpe65 gene transfer protects and restores the function of cones in the Rpe65(-/-) mouse, this study reinforces the therapeutic value of gene therapy for RPE65 deficiencies, suggests a cone-preserving treatment for the retina, and evaluates a potentially effective viral vector for this purpose

Thérapie génique du système nerveux central: Considérations générales sur les vecteurs viraux pour le transfert de gène dans le cerveau

International audienceThe last decade have nourished strong doubts on the beneficial prospects of gene therapy for curing fatal diseases. However, this climate of reservations is currently being transcended by the publication of several successful clinical protocols, restoring confidence in the opportunity of therapeutic gene transfer. A strong sign of this present enthusiasm for gene therapy by clinicians and industrials is the market approval of the therapeutic viral vector Glybera, the first commercial product in Europe of this class of drug. This new field of medicine is particularly attractive when considering therapies for a number of neurological disorders, most of which are desperately waiting for a satisfactory treatment. The central nervous system is indeed a very compliant organ where gene transfer can be stable and successful if provided through an appropriate strategy. The purpose of this review is to present the characteristics of the most efficient virus-derived vectors used by researchers and clinicians to genetically-modify particular cell types or whole regions of the brain. In addition, we discuss major issues regarding side effects such as genotoxicity and immune response associated to the use of these vectorsSuite aux récents succès de divers protocoles thérapeutiques de transfert de gène, notamment appliqués aux pathologies de la rétine, et à la mise sur le marché du Glybera, le premier produit commercial en Europe pour cette classe de médicaments, on observe un regain d'intérêts pour la thérapie génique sur les plans clinique et industriel. Ce nouveau domaine de la médecine expérimentale est particulièrement enthousiasmant si l'on considère que la plupart des maladies neurologiques, attendent désespérément l’apparition d’un traitement satisfaisant. Le système nerveux central est en effet un organe où le transfert de gène peut être stable et réussi s’il est administré selon une stratégie appropriée. L’objectif de cette revue est de présenter les qualités des vecteurs viraux les plus efficaces utilisés actuellement par les chercheurs et les cliniciens pour modifier génétiquement des cellules neurales ou des régions entières du cerveau. Nous abordons également des questions concernant les effets secondaires tels que la génotoxicité et la réponse immunitaire associées à l'utilisation de ces vecteur

Structural and functional connections between the median and the ventrolateral preoptic nucleus. : The MnPO-VLPO pathway

International audienceThe median preoptic nucleus (MnPO) and the ventrolateral preoptic nucleus (VLPO) are two brain structures that contain neurons essential for promoting non-rapid eye movement (NREM) sleep. However, their connections are still largely unknown. Here, we describe for the first time a slice preparation with an oblique coronal slicing angle at 70° from the horizontal in which their connectivity is preserved. Using the in vivo iDISCO method following viral infection of the MnPO or ex vivo biocytin crystal deposition in the MnPO of mouse brain slices, we revealed a strong axonal pathway from the MnPO to the VLPO. Then, to further explore the functionality of these projections, acute 70° slices were placed on multielectrode arrays (MEAs) and electrical stimulations were performed near the MnPO. Recordings of the signals propagation throughout the slices revealed a preferential pathway from the MnPO to the VLPO. Finally, we performed an input-output curve of field responses evoked by stimulation of the MnPO and recorded in the VLPO. We found that field responses were inhibited by GABAA receptor antagonist, suggesting that afferent inputs from the MnPO activate VLPO neuronal networks by disinhibition

IGF-1 exacerbates the neurotoxicity of the mitochondrial inhibitor 3NP in rats

International audienceInsulin-like Growth Factor 1 (IGF-1) has broad-range neuroprotective effects and is a therapeutic candidate for Huntington’s disease (HD). IGF-1 protects striatal neurons from the toxicity of mutated huntingtin $in\ vitro$ and improves neuronal survival $in\ vivo$ in a phenotypic model of HD involving excitotoxic cell death. Because HD is a multifactorial disease, it is important to evaluate the neuroprotective role of IGF-1 in other pathological situations involved in HD progression. We have evaluated the neuroprotective effects of IGF-1 $in\ vivo$, using the 3-nitropropionic acid (3NP) rat model which replicates the mitochondrial dysfunction observed in HD. Continuous intracerebroventricular infusion of recombinant IGF-1 at a low dose (0.025 $\mu$g/h for 5 days) did not alleviate motor impairment and weight loss induced by 3NP treatment. In addition, histological evaluation and quantification of DNA fragmentation evidenced no improvement in neuronal survival. Of interest, we found that a higher concentration of IGF-1 (0.25 $\mu$g/h) resulted in an exacerbation of 3NP toxicity on striatal neurons. These results suggest that intracerebral delivery of IGF-1 may not provide a fully effective therapeutic strategy for HD or other disorders involving mitochondrial impairmen

Insulin growth factor-1 protects against excitotoxicity in the rat striatum

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Lentiviral-mediated gene transfer of brain-derived neurotrophic factor is neuroprotective in a mouse model of neonatal excitotoxic challenge

Excitotoxicity may be a critical factor in the formation of brain lesions associated with cerebral palsy. When injected into the murine neopallium at postnatal day 5, the glutamatergic analog N-methyl-D-aspartate (NMDA) produces transcortical neuronal death and periventricular white matter cysts, which mimic brain damage observed in human term and preterm neonates at risk for developing cerebral palsy. We previously showed that intracerebral injection of brain-derived neurotrophic factor (BDNF) was neuroprotective in this model. Because BDNF does not easily cross the blood-brain barrier, alternative strategies to avoid repeated intracerebral injections of BDNF should be tested, particularly when the goal of such translational research is ultimately to achieve clinical application. The goal of the present study was to assess the protective role of lentiviral-mediated gene transfer of BDNF against excitotoxic lesions induced by NMDA in newborn mice. We first assessed the biological activity of BDNF gene transfer in vitro and determined the efficiency of gene transfer in our in vivo model. We next administered the BDNF-expressing vector by intracerebral injection in neonatal mice, 3 days before inducing NMDA lesions. When compared with a control green fluorescent protein-expressing lentiviral vector, administration of BDNF-expressing vector induced a significant protection of the periventricular white matter and cortical plate against the NMDA-mediated insult. Intraventricular delivery of the BDNF-expressing lentiviral vector was more efficient in terms of neuroprotection than the intraparenchymal route. Altogether, the present study shows that viral-mediated gene transfer of BDNF to newborn mouse brain is feasible and affords significant neuroprotection against an excitotoxic insult

Brain-derived neurotrophic factor-mediated protection of striatal neurons in an excitotoxic rat model of Huntington's disease, as demonstrated by adenoviral gene transfer

International audienceHuntington's disease (HD) is a genetic disorder leading to the degeneration of striatal GABAergic output neurons. No treatment is currently available for this devastating disorder, although several neurotrophic factors, including brain-derived neurotrophic factor (BDNF), have been shown to be beneficial for striatal neuron survival. We analyzed the effect of adenovirusmediated transfer of the BDNF gene in a model of HD. Using a stereological procedure, three groups of rats were given an intrastriatal injection of adenovirus encoding BDNF, ß-galactosidase or sham surgery. Two weeks after treatment, the animals were lesioned with quinolinic acid (QUIN), a toxin which induces striatal neuron death by an excitotoxic process. One month after the lesion, histological study revealed that striatal neurons were protected only in rats treated with the BDNF adenovirus. Volume measurements showed that the QUIN-induced lesions were 55% smaller in the BDNF adenovirus-treated group than in the ßgalactosidase adenovirus-treated group (p<0.05), and the sham-treated group (p<0.05). To determine the survival of striatal GABAergic output neurons after the QUINinduced lesion, we immunostained brain sections with DARPP-32, an antibody specific for striatal output neurons. Prior treatment with the BDNF adenovirus resulted in a cell survival of 64%, whereas that after ßgalactosidase treament was 46% (p<0.05), showing that the BDNF adenovirus protected the striatal neurons. These results indicate that transfer of the BDNF gene is of therapeutic value for Huntington's disease