AAV-mediated Gene Therapy Halts Retinal Degeneration in PDE6ß-deficient Dogs.

We previously reported that subretinal injection of AAV2/5 RK.cpde6ß allowed long-term preservation of photoreceptor function and vision in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency. The present study builds on these earlier findings to provide a detailed assessment of the long-term effects of gene therapy on the spatiotemporal pattern of retinal degeneration in rcd1 dogs treated at 20 days of age. We analyzed the density distribution of the retinal layers and of particular photoreceptor cells in 3.5-year-old treated and untreated rcd1 dogs. Whereas no rods were observed outside the bleb or in untreated eyes, gene transfer halted rod degeneration in all vector-exposed regions. Moreover, while gene therapy resulted in the preservation of cones, glial cells and both the inner nuclear and ganglion cell layers, no cells remained in vector-unexposed retinas, except in the visual streak. Finally, the retinal structure of treated 3.5-year-old rcd1 dogs was identical to that of unaffected 4-month-old rcd1 dogs, indicating near complete preservation. Our findings indicate that gene therapy arrests the degenerative process even if intervention is initiated after the onset of photoreceptor degeneration, and point to significant potential of this therapeutic approach in future clinical trials.

Successful gene therapy in the RPGRIP1-deficient dog: a large model of cone-rod dystrophy.

For the development of new therapies, proof-of-concept studies in large animal models that share clinical features with their human counterparts represent a pivotal step. For inherited retinal dystrophies primarily involving photoreceptor cells, the efficacy of gene therapy has been demonstrated in canine models of stationary cone dystrophies and progressive rod-cone dystrophies but not in large models of progressive cone-rod dystrophies, another important cause of blindness. To address the last issue, we evaluated gene therapy in the retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1)-deficient dog, a model exhibiting a severe cone-rod dystrophy similar to that seen in humans. Subretinal injection of AAV5 (n = 5) or AAV8 (n = 2) encoding the canine Rpgrip1 improved photoreceptor survival in transduced areas of treated retinas. Cone function was significantly and stably rescued in all treated eyes (18-72% of those recorded in normal eyes) up to 24 months postinjection. Rod function was also preserved (22-29% of baseline function) in four of the five treated dogs up to 24 months postinjection. No detectable rod function remained in untreated contralateral eyes. More importantly, treatment preserved bright- and dim-light vision. Efficacy of gene therapy in this large animal model of cone-rod dystrophy provides great promise for human treatment.

Restoration of vision in the pde6ß-deficient dog, a large animal model of rod-cone dystrophy.

Defects in the ß subunit of rod cGMP phosphodiesterase 6 (PDE6ß) are associated with autosomal recessive retinitis pigmentosa (RP), a childhood blinding disease with early retinal degeneration and vision loss. To date, there is no treatment for this pathology. The aim of this preclinical study was to test recombinant adeno-associated virus (AAV)-mediated gene addition therapy in the rod-cone dysplasia type 1 (rcd1) dog, a large animal model of naturally occurring PDE6ß deficiency that strongly resembles the human pathology. A total of eight rcd1 dogs were injected subretinally with AAV2/5RK.cpde6ß (n = 4) or AAV2/8RK.cpde6ß (n = 4). In vivo and post-mortem morphological analysis showed a significant preservation of the retinal structure in transduced areas of both AAV2/5RK.cpde6ß- and AAV2/8RK.cpde6ß-treated retinas. Moreover, substantial rod-derived electroretinography (ERG) signals were recorded as soon as 1 month postinjection (35% of normal eyes) and remained stable for at least 18 months (the duration of the study) in treated eyes. Rod-responses were undetectable in untreated contralateral eyes. Most importantly, dim-light vision was restored in all treated rcd1 dogs. These results demonstrate for the first time that gene therapy effectively restores long-term retinal function and vision in a large animal model of autosomal recessive rod-cone dystrophy, and provide great promise for human treatment.

Intravitreal air tamponade after AAV2 subretinal injection modifies retinal EGFP distribution.

The subretinal injection protocol for the only approved retinal gene therapy (voretigene neparvovec-rzyl) includes air tamponade at the end of the procedure, but its effects on the subretinal bleb have not been described. In the present study, we evaluated the distribution of enhanced green fluorescent protein (EGFP) after subretinal injection of AAV2 in non-human primates (NHP) without (group A = 3 eyes) or with (group B = 3 eyes) air tamponade. The retinal expression of EGFP was assessed 1 month after subretinal injection with in vivo fundus photographs and fundus autofluorescence. In group A (without air), EGFP expression was limited to the area of the initial subretinal bleb. In group B (with air), EGFP was expressed in a much wider area. These data show that the buoyant force of air on the retina causes a wide subretinal diffusion of vector, away from the injection site. In the present paper, we discuss the beneficial and deleterious clinical effects of this finding. Whereas subretinal injection is likely to become more common with the coming of new gene therapies, the effects of air tamponade should be explored further to improve efficacy, reproducibility, and safety of the protocol.

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.

Regulation of retinal function but nonrescue of vision in RPE65-deficient dogs treated with doxycycline-regulatable AAV vectors.

In previous studies, we demonstrated that recombinant adeno-associated virus (rAAV)-mediated gene transfer of the doxycycline (Dox)-regulatable system allows for the regulation of erythropoietin (EPO) expression in the retina of nonhuman primates after intravenous or oral administration of Dox. In addition, it was shown that administrating different amounts of Dox resulted in a dose-response dynamic of transgene expression. Adeno-associated viral gene therapy has raised hope for the treatment of patients with Leber congenital amaurosis, caused by mutations in the retinal pigment epithelium (RPE)-specific gene RPE65. The preliminary results of three clinical trials suggest some improvement in visual function. However, further improvements might be necessary to optimize vision recovery and this means developing vectors able to generate transgene expression at physiological levels. The purpose of this study was to investigate the ability of the Dox-regulatable system to regulate retinal function in RPE65(-/-) Briard dogs. rAAV vectors expressing RPE65 under the control of either the TetOff and TetOn Dox-regulated promoters or the cytomegalovirus (CMV) constitutive promoter were generated and administered subretinally to seven RPE65-deficient dogs. We demonstrate that the induction and deinduction of retinal function, as assessed by electroretinography (ERG), can be achieved using a Dox-regulatable system, but do not lead to any recovery of vision.

Versatile template-directed synthesis of gold nanocages with a predefined number of windows.

Highly symmetrical gold nanocages can be produced with a controllable number of circular windows of either 2, 3, 4, 6 or 12 via an original fabrication route. The synthetic pathway includes three main stages: the synthesis of silica/polystyrene multipod templates, the regioselective seeded growth of a gold shell on the unmasked part of the silica surface and the development of gold nanocages by dissolving/etching the templates. Electron microscopy and tomography provide evidence of the symmetrical features of the as-obtained nanostructures. The optical properties of nanocages with 4 and 12 windows were measured at the single particle level by spatial modulation spectroscopy and correlated with numerical simulations based on finite-element modeling. The new multi-step synthesis approach reported here also allows the synthesis of rattle-like nanostructures through filling of the nanocages with a guest nano-object. With the potential to adjust the chemical composition, size and geometry of both the guest particle and the host cage, it opens new routes towards the fabrication of hollow nanostructures of high interest for a variety of applications including sensing devices, catalytic reactors and biomedicine.

Detection of intact rAAV particles up to 6 years after successful gene transfer in the retina of dogs and primates.

Gene transfer to the retina using recombinant adeno-associated viral (rAAV) vectors has proven to be an effective option for the treatment of retinal degenerative diseases in several animal models and has recently advanced into clinical trials in humans. To date, intracellular trafficking of AAV vectors and subsequent capsid degradation has been studied only in vitro, but the fate of AAV particles in transduced cells following subretinal injection has yet to be elucidated. Using electron microscopy and western blot, we analyzed retinas of one primate and four dogs that had been subretinally injected with AAV2/4, -2/5, or -2/2 serotypes and that displayed efficient gene transfer over several years. We show that intact AAV particles are still present in retinal cells, for up to 6 years after successful gene transfer in these large animals. The persistence of intact vector particles in the target organ, several years postadministration, is totally unexpected and, therefore, represents a new and unanticipated safety issue to consider at a time when gene therapy clinical trials raise new immunological concerns.

Rapid synthesis of ultra-long silver nanowires for high performance transparent electrodes.

By using 1,2-propanediol instead of the classic polyol solvent, ethylene glycol, ultra-long silver nanowires are obtained in only 1 h. These nanowires lead to transparent electrodes with a sheet resistance of 5 Ohms per sq at a transparency of 94%, one of the highest figures of merit for nanowire electrodes ever reported.

The RPGRIP1-deficient dog, a promising canine model for gene therapy.

PURPOSE: To evaluate the RPGRIP1-deficient miniature longhaired dachshund (MLHD) dog as a potential candidate for gene therapy. METHODS: Six RPGRIP1-deficient MLHD dogs from our dog colony have been observed for two years using a variety of noninvasive procedures. These included bilateral full-field electroretinograms (ERG) to evaluate retinal function, fundus photographs to evaluate retinal vascularization, and optical coherence tomographs (OCT) to evaluate retinal thickness. We also performed histological examination of hematoxylin- and eosin-stained retinal sections as well as sections labeled in situ by the terminal dUTP nick end labeling (TUNEL) method. RESULTS: ERG findings showed that as early as 2 months of age, cone function was lost while rod function was preserved. However, by 9 months of age, both cone and rod functions could not be detected. Functional visual assessment based on the ability to avoid obstacles showed that vision was retained up to the age of 11 months. Both OCT and histopathology studies revealed a progressive thinning of the outer nuclear layer (ONL) over the first 2 years of age. TUNEL labeling identified apoptotic photoreceptor cell death as the cause of this thinning of the ONL. CONCLUSIONS: A treatment strategy should consist in initiating gene therapy as early as possible after birth to prevent or delay the loss of rod function. In the MLHD, successful subretinal delivery of a therapeutic vector is feasible at 2 months of age and may prevent or delay the loss of rod function.

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.

Increasing Silver Nanowire Network Stability through Small Molecule Passivation.

Silver nanowire (AgNW) transparent electrodes show promise as an alternative to indium tin oxide (ITO). However, these nanowire electrodes degrade in air, leading to significant resistance increases. We show that passivating the nanowire surfaces with small organic molecules of 11-mercaptoundecanoic acid (MUA) does not affect electrode transparency contrary to typical passivation films, and is inexpensive and simple to deposit. The sheet resistance of a 32 nm diameter silver nanowire network coated with MUA increases by only 12% over 120 days when exposed to atmospheric conditions but kept in the dark. The increase is larger when exposed to daylight (588%), but is still nearly two orders of magnitude lower than the resistance increase of unpassivated networks. The difference between the experiments performed under daylight versus the dark exemplifies the importance of testing passivation materials under light exposure.

Outer Plexiform Layer Structures Are Not Altered Following AAV-Mediated Gene Transfer in Healthy Rat Retina.

Ocular gene therapy approaches have been developed for a variety of different diseases. In particular, clinical gene therapy trials for RPE65 mutations, X-linked retinoschisis, and choroideremia have been conducted at different centers in recent years, showing that adeno-associated virus (AAV)-mediated gene therapy is safe, but limitations exist as to the therapeutic benefit and long-term duration of the treatment. The technique of vector delivery to retinal cells relies on subretinal injection of the vector solution, causing a transient retinal detachment. Although retinal detachments are known to cause remodeling of retinal neuronal structures as well as significant cell loss, the possible effects of this short-term therapeutic retinal detachment on retinal structure and circuitry have not yet been studied in detail. In this study, retinal morphology and apoptotic status were examined in healthy rat retinas following AAV-mediated gene transfer via subretinal injection with AAV2/5.CMV.d2GFP or sham injection with fluorescein. Outer plexiform layer (OPL) morphology was assessed by immunohistochemical labeling, laser scanning confocal microscopy, and electron microscopy. The number of synaptic contacts in the OPL was quantified after labeling with structural markers. To assess the apoptotic status, inflammatory and pro-apoptotic markers were tested and TUNEL assay for the detection of apoptotic nuclei was performed. Pre- and postsynaptic structures in the OPL, such as synaptic ribbons or horizontal and bipolar cell processes, did not differ in size or shape in injected versus non-injected areas and control retinas. Absolute numbers of synaptic ribbons were not altered. No signs of relevant gliosis were detected. TUNEL labeling of retinal cells did not vary between injected and non-injected areas, and apoptosis-inducing factor was not delocalized to the nucleus in transduced areas. The neuronal circuits in the OPL of healthy rat retinas undergoing AAV-mediated gene transfer were not altered by the temporary retinal detachment caused by subretinal injection, the presence of viral particles, or the expression of green fluorescent protein as a transgene. This observation likely requires further investigations in the dog model for RPE65 deficiency in order to determine the impact of RPE65 transgene expression on diseased retinas in animals and men.

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.

Fisioterapia extra-hospitalar durante a pandemia: a visão e o posicionamento dos profissionais / Fisioterapia extrahospitalaria durante la pandemia: la visión y el posicionamiento de los profesionales / Out-of-hospital physical therapy during pandemic: the vision and positioning of professionals

RESUMO A pandemia da COVID-19 provocou momentos atípicos para a população. A fim de promover medidas de controle da disseminação viral, decretos emitidos pelos diversos níveis governamentais indicaram serviços essenciais e não essenciais que poderiam permanecer em funcionamento no Brasil durante determinado período. A fisioterapia extra-hospitalar foi considerada não essencial. O artigo tem como objetivo verificar se a fisioterapia no âmbito da atuação extra-hospitalar é considerada um serviço essencial, na visão de fisioterapeutas, durante os períodos de crises humanitárias, a exemplo da pandemia causada pela COVID-19, e identificar os tipos de procedimentos assistenciais executados. Para isso, foi realizada pesquisa transversal, quantitativa e descritiva do tipo survey com análise descritiva. Realizada por meio de um questionário eletrônico publicado nos sites dos Conselhos Regionais de Fisioterapia e Terapia Ocupacional (Crefito) do Paraná, de Santa Catarina, e do Rio Grande do Sul (respectivamente, Crefito 8, 10 e 5). Nos resultados, foi observado que 78% dos voluntários são do sexo feminino, sendo que 44% estão registrados no Crefito 8, 40% são fisioterapeutas do Crefito 5, 16% são registrados no Crefito 10 e 100% da amostra considerou a fisioterapia extra-hospitalar um serviço essencial. Em relação ao grau de formação, 70% dos profissionais que responderam possuem pós-graduação lato-sensu e 54% atuam em estabelecimentos privados. Durante o decreto de serviços essenciais, 56% dos profissionais não atuaram. Com isso, conclui-se que a fisioterapia extra-hospitalar é essencial em crises pandêmicas. Além de dar início e proporcionar continuidade ao tratamento do paciente, evita visitas desnecessárias aos hospitais.

RESUMEN La pandemia del COVID-19 provocó momentos atípicos para la población de todo el mundo. Con el fin de promover medidas para evitar la propagación del virus por Brasil, los decretos emitidos por los gobiernos indicaron servicios esenciales y no esenciales que podrían permanecer en funcionamiento por determinado periodo. La fisioterapia extrahospitalaria se consideró no esencial. Este artículo tiene por objetivo verificar si los profesionales consideran la fisioterapia en el contexto de la acción extrahospitalaria como un servicio esencial durante periodos de crisis humanitaria, como la pandemia provocada por el COVID-19, así como identificar los tipos de procedimientos asistenciales realizados. Para ello, se realizó una investigación transversal, cuantitativa y descriptiva, del tipo encuesta, con análisis descriptivo. Se usó un cuestionario electrónico publicado en los sitios web de los Consejos Regionales de Fisioterapia y Terapia Ocupacional (Crefito) de Paraná, de Santa Catarina y de Rio Grande do Sul (Crefito 8, 10 y 5, respectivamente). Los resultados indican que el 78% de los voluntarios son mujeres, el 44% están registrados en Crefito 8, el 40% son fisioterapeutas en Crefito 5, el 16% están registrados en Crefito 10, y el 100% de la muestra considera la fisioterapeuta extrahospitalaria un servicio esencial. En cuanto al nivel educativo, el 70% de los profesionales que respondieron tiene posgrado lato-sensu y el 54% trabajan en establecimientos privados. Durante el decreto sobre servicios esenciales, el 56% de los profesionales no trabajaban. Esto permite concluir que la fisioterapia extrahospitalaria es fundamental en crisis pandémicas. Además de iniciar y dar continuidad al tratamiento del paciente, evita que este vaya a hospitales sin necesidad.

ABSTRACT The COVID-19 pandemic caused atypical moments for the population. In Brazil, to promote measures to control viral dissemination, Decrees issued by several government levels indicated the essential and non-essential services that could remain in operation during a certain period. Out-of-hospital physical therapy was considered nonessential. This article aims to verify whether physical therapists consider the practice of out-of-hospital physical therapy as an essential service during periods of humanitarian crises, such as the COVID-19 pandemic. Additionally, we aim to identify the types of care procedures performed during this period. We performed a cross-sectional, quantitative, and descriptive survey with descriptive analysis, conducted by an electronic questionnaire published on the websites of the Regional Councils of Physical Therapy and Occupational Therapy (CREFITO's) of Paraná - CREFITO 8, Santa Catarina - CREFITO 10, and Rio Grande do Sul - CREFITO 5. 78% of the volunteers are female, and 44% are registered in CREFITO 8, 40% are physical therapist of CREFITO 5, 16% are registered in CREFITO 10, and 100% of the sample considered out-of-hospital physical therapy an essential service. Regarding the schooling level, 70% have a graduate degree and 54% work in private establishments. During the decree of essential services, 56% of the professionals did not practice. Out-of-hospital physical therapy is essential in pandemic crises. In addition to providing initiation and continuity to patient treatment, it avoids unnecessary visits to hospitals.

MSRV envelope protein is a potent, endogenous and pathogenic agonist of human toll-like receptor 4: Relevance of GNbAC1 in multiple sclerosis treatment.

Multiple sclerosis associated retrovirus envelope protein (MSRV-Env) was repeatedly detected in brain lesions and blood of multiple sclerosis (MS) patients. We performed the first pharmacological characterisation of MSRV-Env on recombinant and native human TLR4. MSRV-Env is a full and highly potent TLR4 agonist of endogenous origin. MSRV-Env induces TLR4-dependent pro-inflammatory stimulation of immune cells in vitro and in vivo, and impairs oligodendrocytes precursor cells differentiation to myelinating oligodendrocytes. MSRV-Env may play a role in chronic inflammation and impaired remyelination in MS. GNbAC1, a selective monoclonal antibody, antagonizes MSRV-Env pathogenic effects and represents an innovative therapeutic approach of MS.

Human Endogenous Retrovirus and Neuroinflammation in Chronic Inflammatory Demyelinating Polyradiculoneuropathy.

BACKGROUND: Human endogenous retroviruses HERV-W encode a pro-inflammatory protein, named MSRV-Env from its original identification in Multiple Sclerosis. Though not detected in various neurological controls, MSRV-Env was found in patients with chronic inflammatory demyelinating polyradiculoneuropathies (CIDPs). This study investigated the expression of MSRV in CIDP and evaluated relevant MSRV-Env pathogenic effects. METHODS: 50 CIDP patients, 19 other neurological controls (ONDs) and 65 healthy blood donors (HBDs) were recruited from two different countries. MSRV-env and -pol transcripts, IL6 and CXCL10 levels were quantified from blood samples. MSRV-Env immunohistology was performed in distal sensory nerves from CIDP and neurological controls biopsies. MSRV-Env pathogenic effects and mode of action were assayed in cultured primary human Schwann cells (HSCs). FINDINGS: In both cohorts, MSRV-env and -pol transcripts, IL6 positivity prevalence and CXCL10 levels were significantly elevated in CIDP patients when compared to HBDs and ONDs (statistically significant in all comparisons). MSRV-Env protein was detected in Schwann cells in 5/7 CIDP biopsies. HSC exposed to or transfected with MSRV-env presented a strong increase of IL6 and CXCL10 transcripts and protein secretion. These pathogenic effects on HSC were inhibited by GNbAC1, a highly specific and neutralizing humanized monoclonal antibody targeting MSRV-Env. INTERPRETATION: The present study showed that MSRV-Env may trigger the release of critical immune mediators proposed as instrumental factors involved in the pathophysiology of CIDP. Significant MSRV-Env expression was detected in a significant proportion of patients with CIDP, in which it may play a role according to its presently observed effects on Schwann cells along with previously known effects on immune cells. Experimental results also suggest that a biomarker-driven therapeutic strategy targeting this protein with a neutralizing antibody such as GNbAC1 may offer new perspectives for treating CIDP patients with positive detection of MSRV-Env expression. FUNDING: Geneuro-Innovation, France.

Vitrectomy Before Intravitreal Injection of AAV2/2 Vector Promotes Efficient Transduction of Retinal Ganglion Cells in Dogs and Nonhuman Primates.

Recombinant adeno-associated virus (AAV) has emerged as a promising vector for retinal gene delivery to restore visual function in certain forms of inherited retinal dystrophies. Several studies in rodent models have shown that intravitreal injection of the AAV2/2 vector is the optimal route for efficient retinal ganglion cell (RGC) transduction. However, translation of these findings to larger species, including humans, is complicated by anatomical differences in the eye, a key difference being the comparatively smaller volume of the vitreous chamber in rodents. Here, we address the role of the vitreous body as a potential barrier to AAV2/2 diffusion and transduction in the RGCs of dogs and macaques, two of the most relevant preclinical models. We intravitreally administered the AAV2/2 vector carrying the CMV-eGFP reporter cassette in dog and macaque eyes, either directly into the vitreous chamber or after complete vitrectomy, a surgical procedure that removes the vitreous body. Our findings suggest that the vitreous body appears to trap the injected vector, thus impairing the diffusion and transduction of AAV2/2 to inner retinal neurons. We show that vitrectomy before intravitreal vector injection is an effective means of overcoming this physical barrier, improving the transduction of RGCs in dog and macaque retinas. These findings support the use of vitrectomy in clinical trials of intravitreal gene transfer techniques targeting inner retinal neurons.

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.

The 5-HT2A receptor is widely distributed in the rat spinal cord and mainly localized at the plasma membrane of postsynaptic neurons.

Serotonin (5-HT) plays a major role at the spinal level by modulating most spinal functions through several receptor subtypes including the 5-HT2A receptor. To gain further insight into the cellular role of this receptor, we performed an immunocytochemical study of 5-HT2A receptors in the rat spinal cord, at light and electron microscope levels. The results showed that 5-HT2A receptors were widely distributed in the spinal cord at all segmental levels. Immunolabeling was particularly dense in lamina IX and in the dorsal horn lamina IIi. Immunoreactive cell bodies were numerous in lamina IX, where many but not all motoneurons were labeled, as shown by double labeling with choline acetyltransferase antibodies. Stained cell bodies were also observed in the gray matter. The study at the ultrastructural level focused on the lumbar dorsal horn (laminae I-II) and ventral horn (lamina IX). At both levels, 5-HT2A immunoreactivity was mainly postsynaptic on dendrites and cell bodies. However, a little presynaptic labeling was also observed in axon and axon terminals, some of them containing large granular vesicles attesting to their peptidergic nature. The main result of our study was the "nonsynaptic" plasma membrane localization of 5-HT2A receptors covering a large surface of cell bodies and dendrites, suggesting a paracrine form of action of serotonin. These observations are consistent with a double role (pre- and postsynaptic) for serotonin on these receptors on various cellular targets.