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Divya Ail, Hugo Malki, Emilia A Zin, Deniz Dalkara Sorbonne Université, INSERM, CNRS, Department of Therapeutics, Institut de la Vision, Paris, 75012, FranceCorrespondence: Divya Ail, Department of Therapeutics, Institut de la Vision, 17 Rue Moreau, Paris, 75012, France, Tel +33 153462648, Email divya.ail@inserm.frAbstract: Owing to their small size and safety profiles, adeno-associated viruses (AAVs) have become the vector of choice for gene therapy applications in the retina. In addition to the naturally occurring AAVs, several engineered variants with enhanced properties are being developed for experimental and therapeutic applications. Nonetheless, there are still some challenges impeding successful application of AAVs for a broader range of retinal gene therapies. The small size of AAV particles ensures efficient tissue transduction but also limits the packaging capacity to a few kilobases. Further, AAV’s ability to cross retinal barriers is still an obstacle to pan-retinal transduction of the outer retina with tolerable doses. Lastly, despite overall safety, there have been recent reports of immune responses to AAVs in the eye. Hence, evaluation and prediction of immune responses to AAVs has come to be considered an integral part of future clinical success. This review focuses on the use of AAV in clinical trials for retinal diseases, and discusses developments of variants and novel strategies to overcome immune responses to AAVs.Keywords: retinal gene therapy, adeno-associated virus, AAV, clinical trials, capsid variants, immune responses

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.

Nogo-A is a well-known myelin-enriched inhibitory protein for axonal growth and regeneration in the central nervous system (CNS). Besides oligodendrocytes, our previous data revealed that Nogo-A is also expressed in subpopulations of neurons including retinal ganglion cells, in which it can have a positive role in the neuronal growth response after injury, through an unclear mechanism. In the present study, we analyzed the opposite roles of glial versus neuronal Nogo-A in the injured visual system. To this aim, we created oligodendrocyte (Cnp-Cre+/−xRtn4/Nogo-Aflox/flox) and neuron-specific (Thy1-Cretg+xRtn4flox/flox) conditional Nogo-A knock-out (KO) mouse lines. Following complete intraorbital optic nerve crush, both spontaneous and inflammation-mediated axonal outgrowth was increased in the optic nerves of the glia-specific Nogo-A KO mice. In contrast, neuron-specific deletion of Nogo-A in a KO mouse line or after acute gene recombination in retinal ganglion cells mediated by adeno-associated virus serotype 2.Cre virus injection in Rtn4flox/flox animals decreased axon sprouting in the injured optic nerve. These results therefore show that selective ablation of Nogo-A in oligodendrocytes and myelin in the optic nerve is more effective at enhancing regrowth of injured axons than what has previously been observed in conventional, complete Nogo-A KO mice. Our data also suggest that neuronal Nogo-A in retinal ganglion cells could participate in enhancing axonal sprouting, possibly by cis-interaction with Nogo receptors at the cell membrane that may counteract trans-Nogo-A signaling. We propose that inactivating Nogo-A in glia while preserving neuronal Nogo-A expression may be a successful strategy to promote axonal regeneration in the CNS. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Gene augmentation therapy is a promising treatment for incurable, blinding inherited retinal diseases, and intravitreal delivery is being studied as a safe alternative to subretinal injections. Adeno-Associated Viruses (AAV) are commonly-used vectors for ocular gene augmentation therapy. Naturally occurring pre-operative exposure and infection with AAV could result in presence of neutralizing antibodies (NAB's) in patients' serum, and may affect the safety and efficacy of treatment. Our aim was to characterize the humoral response against AAV pre- and post-intravitreal delivery of AAV2.7m8 vectors in a naturally-occurring sheep model of CNGA3 achromatopsia. Serial serum neutralization assays were performed to screen sheep for pre-exiting anti-AAV2 NAB's, and to assess the effect of intravitreal AAV2.7m8 injection on post-operative NAB titers and intraocular inflammation in sheep. The effect of viral dose and transgene type were also assessed. Serological screening revealed pre-operative seropositivity in 21.4% of animals, with age being a risk factor for the presence of anti-AAV2 NAB's. NAB titers increased following intravitreal AAV administration in the majority of sheep. There was no significant difference in the degree of post-operative serum neutralization between pre-operatively seronegative sheep and those with pre-existing antibodies. However, only sheep with pre-existing antibodies presented with signs of post-operative inflammation. We conclude that pre-existing anti-AAV2 NAB's do not affect the level of post-operative NAB titers; however, they increase the risk of post-operative ocular inflammation. Our results could have implications for the management of AAV-mediated ocular gene therapies, a technology being increasingly studied and used in patients., Competing Interests: Competing interests M. Ross, none; K. Sade, none; A. Obolensky none; E. Averbukh, none; M. Desrosiers, None A. Rosov, none; H. Dvir, none; E. Gootwine, none; E. Banin, none; D. Dalkara reports grants from Foundation Fighting Blindness, USA and European Research Council, during the conduct of the study; DD is a co-inventor on patent #9193956 (Adeno-associated virus virions with variant capsid and methods of use thereof), with royalties paid to Adverum Biotechnologies and on pending patent applications on noninvasive methods to target cone photoreceptors (EP17306429.6 and EP17306430.4) licensed to Gamut Tx now Sparing- Vision. DD also has personal financial interests in Tenpoint Tx. and SparingVision, outside the scope of the submitted work; R. Ofri, none. Ethical approval Experimental protocols were approved by the Agricultural Research Organization, Volcani Center, Animal Experimentation Ethics Committee, Permit 643/16, (© 2024. The Author(s).)

Genome editing by CRISPR-Cas holds promise for the treatment of retinal dystrophies. For therapeutic gene editing, transient delivery of CRISPR-Cas9 is preferable to viral delivery which leads to long-term expression with potential adverse consequences. Cas9 protein and its guide RNA, delivered as ribonucleoprotein (RNP) complexes, have been successfully delivered into the retinal pigment epithelium in vivo . However, the delivery into photoreceptors, the primary focus in retinal dystrophies, has not been achieved. Here, we investigate the feasibility of direct RNP delivery into photoreceptors and retinal pigment epithelium cells. We demonstrate that Cas9 or adenine-base editors complexed with guide RNA, can enter retinal cells without the addition of any carrier compounds. Once in the retinal cells, editing rates vary based on the efficacy of the guide RNA and the specific location edited within the genes. Cas9 RNP delivery at high concentrations, however, leads to outer retinal toxicity. This underscores the importance of improving delivery efficiency for potential therapeutic applications in the future., Competing Interests: D.D. is a co-inventor on patent #9193956 (Adeno-associated virus virions with variant capsid and methods of use thereof), with royalties paid to Adverum Biotechnologies and on pending patent applications on noninvasive methods to target cone photoreceptors (EP17306429.6 and EP17306430.4) licensed to Gamut Tx now SparingVision. D.D. also has personal financial interests in Tenpoint Tx. and SparingVision, outside the scope of the submitted work., (© 2024 The Author(s).)

After more than two decades of basic research and preclinical studies, adeno-associated virus (AAV)-mediated gene transfer has been tested successfully in clinical trials to treat inherited retinal diseases. Despite the eye's immune-privileged status, some patients display inflammatory events requiring the use of corticoids as an adjunct treatment which led us to question the immune consequences of a subretinal AAV administration. We first characterized anti-transgene immune responses induced in the periphery by injecting increasing doses of AAV8 encoding reporter proteins fused with the HY male antigen into the subretinal space of female C57BL/6 and rd10 mice. Transgene expression was monitored over time with bioluminescence imaging, and T cell immune responses in the spleen were analyzed by IFNγ ELISpot and cytokine multiplex assays. Our data show that AAV8 injections cause pro-inflammatory T cell immune response against the transgene product correlated with the transgene expression level at 2.10 9 vg and above. In addition, co-injection of immunodominant peptides from the transgene product, along with AAV8, modulates the immune response at all AAV doses tested. Taken together, our data suggest that injection of AAV8 in the subretinal space induces pro-inflammatory peripheral T cell responses to the transgene product that can be modulated by the subretinal-associated immune inhibition mechanism.

A deficiency in the shortest dystrophin-gene product, Dp71, is a pivotal aggravating factor for intellectual disabilities in Duchenne muscular dystrophy (DMD). Recent advances in preclinical research have achieved some success in compensating both muscle and brain dysfunctions associated with DMD, notably using exon skipping strategies. However, this has not been studied for distal mutations in the DMD gene leading to Dp71 loss. In this study, we aimed to restore brain Dp71 expression in the Dp71-null transgenic mouse using an adeno-associated virus (AAV) administrated either by intracardiac injections at P4 (ICP4) or by bilateral intracerebroventricular (ICV) injections in adults. ICP4 delivery of the AAV9-Dp71 vector enabled the expression of 2 to 14% of brain Dp71, while ICV delivery enabled the overexpression of Dp71 in the hippocampus and cortex of adult mice, with anecdotal expression in the cerebellum. The restoration of Dp71 was mostly located in the glial endfeet that surround capillaries, and it was associated with partial localization of Dp71-associated proteins, α1-syntrophin and AQP4 water channels, suggesting proper restoration of a scaffold of proteins involved in blood-brain barrier function and water homeostasis. However, this did not result in significant improvements in behavioral disturbances displayed by Dp71-null mice. The potential and limitations of this AAV-mediated strategy are discussed. This proof-of-concept study identifies key molecular markers to estimate the efficiencies of Dp71 rescue strategies and opens new avenues for enhancing gene therapy targeting cognitive disorders associated with a subgroup of severely affected DMD patients.

Forward genetic screens of mutated variants are a versatile strategy for protein engineering and investigation, which has been successfully applied to various studies like directed evolution (DE) and deep mutational scanning (DMS). While next-generation sequencing can track millions of variants during the screening rounds, the vast and noisy nature of the sequencing data impedes the estimation of the performance of individual variants. Here, we propose ACIDES that combines statistical inference and in-silico simulations to improve performance estimation in the library selection process by attributing accurate statistical scores to individual variants. We tested ACIDES first on a random-peptide-insertion experiment and then on multiple public datasets from DE and DMS studies. ACIDES allows experimentalists to reliably estimate variant performance on the fly and can aid protein engineering and research pipelines in a range of applications, including gene therapy., (© 2023. The Author(s).)

Most inherited retinal dystrophies display progressive photoreceptor cell degeneration leading to severe visual impairment. Optogenetic reactivation of inner retinal neurons is a promising avenue to restore vision in retinas having lost their photoreceptors. Expression of optogenetic proteins in surviving ganglion cells, the retinal output, allows them to take on the lost photoreceptive function. Nonetheless, this creates an exclusively ON retina by expression of depolarizing optogenetic proteins in all classes of ganglion cells, whereas a normal retina extracts several features from the visual scene, with different ganglion cells detecting light increase (ON) and light decrease (OFF). Refinement of this therapeutic strategy should thus aim at restoring these computations. Here we used a vector that targets gene expression to a specific interneuron of the retina called the AII amacrine cell. AII amacrine cells simultaneously activate the ON pathway and inhibit the OFF pathway. We show that the optogenetic stimulation of AII amacrine cells allows restoration of both ON and OFF responses in the retina, but also mediates other types of retinal processing such as sustained and transient responses. Targeting amacrine cells with optogenetics is thus a promising avenue to restore better retinal function and visual perception in patients suffering from retinal degeneration., Competing Interests: D.D. reports grants from Foundation Fighting Blindness, USA and European Research Council, during the conduct of the study; D.D. is a co-inventor on patent #9193956 (Adeno-associated virus virions with variant capsid and methods of use thereof), with royalties paid to Adverum Biotechnologies. D.D. also has personal financial interests in Tenpoint Tx. and SparingVision, outside the submitted work. D.D., H.K., O.M., F.T., and E.O. are inventors on pending patent applications on methods to target A2 amacrine cells related to this work., (© 2023 The Authors.)

The anatomical differences between the retinas of humans and most animal models pose a challenge for testing novel therapies. Nonhuman primate (NHP) retina is anatomically closest to the human retina. However, there is a lack of relevant NHP models of retinal degeneration (RD) suitable for preclinical studies. To address this unmet need, we generated three distinct inducible cynomolgus macaque models of RD. We developed two genetically targeted strategies using optogenetics and CRISPR-Cas9 to ablate rods and mimic rod-cone dystrophy. In addition, we created an acute model by physical separation of the photoreceptors and retinal pigment epithelium using a polymer patch. Among the three models, the CRISPR-Cas9-based approach was the most advantageous model in view of recapitulating disease-specific features and its ease of implementation. The acute model, however, resulted in the fastest degeneration, making it the most relevant model for testing end-stage vision restoration therapies such as stem cell transplantation.

Remote and precisely controlled activation of the brain is a fundamental challenge in the development of brain-machine interfaces for neurological treatments. Low-frequency ultrasound stimulation can be used to modulate neuronal activity deep in the brain, especially after expressing ultrasound-sensitive proteins. But so far, no study has described an ultrasound-mediated activation strategy whose spatiotemporal resolution and acoustic intensity are compatible with the mandatory needs of brain-machine interfaces, particularly for visual restoration. Here we combined the expression of large-conductance mechanosensitive ion channels with uncustomary high-frequency ultrasonic stimulation to activate retinal or cortical neurons over millisecond durations at a spatiotemporal resolution and acoustic energy deposit compatible with vision restoration. The in vivo sonogenetic activation of the visual cortex generated a behaviour associated with light perception. Our findings demonstrate that sonogenetics can deliver millisecond pattern presentations via an approach less invasive than current brain-machine interfaces for visual restoration., (© 2023. The Author(s).)

Since their discovery over 55 years ago, adeno-associated virus (AAV) vectors have become powerful tools for experimental and therapeutic in vivo gene delivery, particularly in the retina. Increasing knowledge of AAV structure and biology has propelled forward the development of engineered AAV vectors with improved abilities for gene delivery. However, major obstacles to safe and efficient therapeutic gene delivery remain, including tropism, inefficient and untargeted gene delivery, and limited carrying capacity. Additional improvements to AAV vectors will be required to achieve therapeutic benefit while avoiding safety issues. In this review, we provide an overview of recent methods for engineering-enhanced AAV capsids, as well as remaining challenges that must be overcome to achieve optimized therapeutic gene delivery in the eye., (Copyright © 2023 Cold Spring Harbor Laboratory Press; all rights reserved.)

Gene therapy is a potential cure for several inherited retinal dystrophies, and adeno-associated virus (AAV) has emerged as a vector of choice for therapeutic gene delivery to the retina. However, prior exposure to AAVs can cause a humoral immune response resulting in the presence of antibodies in the serum, which can subsequently interfere with the AAV-mediated gene therapy. The antibodies bind specifically to a serotype but often display broad cross-reactivity. A subset of these antibodies called neutralizing antibodies (NABs) can render the AAV inactive, thereby reducing the efficacy of the therapy. The preexisting NAB levels against different serotypes vary by species, and these variations need to be considered while designing studies. Since large animals often serve as preclinical models to test gene therapies, in this review we compile studies reporting preexisting NABs against commonly used AAV serotypes in humans and large animal models and discuss strategies to deal with NABs., (© 2023. The Author(s), under exclusive license to Springer Nature Switzerland AG.)

The recent approval of voretigene neparvovec (Luxturna®) for patients with biallelic RPE65 mutation-associated inherited retinal dystrophy with viable retinal cells represents an important step in the development of ocular gene therapies. Herein, we review studies investigating the episomal persistence of different recombinant adeno-associated virus (rAAV) vector genomes and the preclinical and clinical evidence of long-term effects of different RPE65 gene replacement therapies. A targeted review of articles published between 1974 and January 2021 in Medline®, Embase®, and other databases was conducted, followed by a descriptive longitudinal analysis of the clinical trial outcomes of voretigene neparvovec. Following an initial screening, 14 publications examining the episomal persistence of different rAAV genomes and 71 publications evaluating gene therapies in animal models were included. Viral genomes were found to persist for at least 22 months (longest study follow-up) as transcriptionally active episomes. Treatment effects lasting almost a decade were reported in canine disease models, with more pronounced effects the earlier the intervention. The clinical trial outcomes of voretigene neparvovec are consistent with preclinical findings and reveal sustained results for up to 7.5 years for the full-field light sensitivity threshold test and 5 years for the multi-luminance mobility test in the Phase I and Phase III trials, respectively. In conclusion, the therapeutic effect of voretigene neparvovec lasts for at least a decade in animal models and 7.5 years in human subjects. Since retinal cells can retain functionality over their lifetime after transduction, these effects may be expected to last even longer in patients with a sufficient number of outer retinal cells at the time of intervention., (© 2022 The Author(s). Published by S. Karger AG, Basel.)

Accumulation of the microtubule-associated protein Tau is linked to neuronal cell death in tauopathies, but how intraneuronal Tau levels are regulated in health and disease remains unclear. Here, we show that conditional inactivation of the trafficking adaptor protein Numb in retinal ganglion cells (RGCs) increases Tau levels and leads to axonal blebbing, which is followed by neuronal cell loss in aged mice. In the TauP301S mouse model of tauopathy, conditional inactivation of Numb in RGCs and spinal motoneurons accelerates neurodegeneration, and loss of Numb in motoneurons also leads to precocious hindlimb paralysis. Conversely, overexpression of the long isoform of Numb (Numb-72) decreases intracellular Tau levels and reduces axonal blebbing in TauP301S RGCs, leading to improved electrical activity in cultured neurons and improves performance in a visually guided behavior test in vivo. These results uncover Numb as a key regulator of intracellular Tau levels and identify Numb-72 as a potential therapeutic factor for tauopathies.

Inherited retinal diseases (IRDs) are a leading cause of blindness in industrialized countries, and gene therapy is quickly becoming a viable option to treat this group of diseases. Gene replacement using a viral vector has been successfully applied and advanced to commercial use for a rare group of diseases. This, and the advances in gene editing, are paving the way for the emergence of a new generation of therapies that use CRISPR-Cas9 to edit mutated genes in situ. These CRISPR-based agents can be delivered to the retina as transgenes in a viral vector, unpackaged transgenes or as proteins or messenger RNA using non-viral vectors. Although the eye is considered to be an immune-privileged organ, studies in animals, as well as evidence from clinics, have concluded that ocular gene therapies elicit an immune response that can under certain circumstances result in inflammation. In this review, we evaluate studies that have reported on pre-existing immunity, and discuss both innate and adaptive immune responses with a specific focus on immune responses to gene editing, both with non-viral and viral delivery in the ocular space. Lastly, we discuss approaches to prevent and manage the immune responses to ensure safe and efficient gene editing in the retina.

We developed a multi-unit microscope for all-optical inter-layers circuits interrogation. The system performs two-photon (2P) functional imaging and 2P multiplexed holographic optogenetics at axially distinct planes. We demonstrated the capability of the system to map, in the mouse retina, the functional connectivity between rod bipolar cells (RBCs) and ganglion cells (GCs) by activating single or defined groups of RBCs while recording the evoked response in the GC layer with cell-type specificity and single-cell resolution. We then used a logistic model to probe the functional connectivity between cell types by deriving the "cellular receptive field" describing how RBCs impact each GC type. With the capability to simultaneously image and control neuronal activity at axially distinct planes, the system enables a precise interrogation of multi-layered circuits. Understanding this information transfer is a promising avenue to dissect complex neural circuits and understand the neural basis of computations., Competing Interests: Anurag Agrawal is an employee of Double Helix Optics, Inc., (© 2022 The Author(s).)

Inherited retinal dystrophies (IRDs) are a heterogeneous group of diseases that affect more than 2 million people worldwide. Gene therapy (GT) has emerged as an exciting treatment modality with the potential to provide long-term benefit to patients. Today, gene addition is the most straightforward GT for autosomal recessive IRDs. However, there are three scenarios where this approach falls short. First, in autosomal dominant diseases caused by gain-of-function or dominant-negative mutations, the toxic mutated protein needs to be silenced. Second, a number of IRD genes exceed the limited carrying capacity of adeno-associated virus vectors. Third, there are still about 30% of patients with unknown mutations. In the first two contexts, precise editing tools, such as CRISPR-Cas9, base editors, or prime editors, are emerging as potential GT solutions for the treatment of IRDs. Here, we review gene editing tools based on CRISPR-Cas9 technology that have been used in vivo and the recent first-in-human application of CRISPR-Cas9 in an IRD.

Positive clinical outcomes in adeno-associated virus (AAV)-mediated retinal gene therapy have often been attributed to the low immunogenicity of AAVs and immune privilege of the eye. However, several recent studies have shown potential for inflammatory responses. The current understanding of the factors contributing to inflammation, such as the pre-existence of serum antibodies against AAVs and their contribution to increases in antibody levels post-injection, is incomplete. The parameters that regulate the generation of new antibodies in response to the AAV capsid or transgene after intraocular injections are also insufficiently described. This study is a retrospective analysis of the pre-existing serum antibodies in correlation with changes in antibody levels after intraocular injections of AAV in non-human primates (NHPs) of the species Macaca fascicularis . In NHP serums, we analyzed the binding antibody (BAB) levels and a subset of these called neutralizing antibodies (NABs) that impede AAV transduction. We observed significantly higher pre-existing serum BABs against AAV8 compared with other serotypes and a dose-dependent increase in BABs and NABs in the serums collected post-injection, irrespective of the serotype or the mode of injection. Lastly, we were able to demonstrate a correlation between the serum BAB levels with clinical grading of inflammation and levels of transgene expression., Competing Interests: D.D. is an inventor on a patent of AAV virions with variant capsid and methods of use thereof with royalties paid to Adverum Biotechnologies (WO2012145601 A2) and on patent applications on non-invasive methods to target cone photoreceptors (EP17306429.6 and EP17306430.4) licensed to Gamut Tx (now SparingVision). D.D. is a founder of Gamut Tx (now SparingVision). All other authors declare no competing interests., (© 2022 The Authors.)

Inherited and age-related retinal degeneration is the hallmark of a large group of heterogeneous diseases and is the main cause of untreatable blindness today. Genetic factors play a major pathogenic role in retinal degenerations for both monogenic diseases (such as retinitis pigmentosa) and complex diseases with established genetic risk factors (such as age-related macular degeneration). Progress in genotyping techniques and back of the eye imaging are completing our understanding of these diseases and their manifestations in patient populations suffering from retinal degenerations. It is clear that whatever the genetic cause, the majority of vision loss in retinal diseases results from the loss of photoreceptor function. The timing and circumstances surrounding the loss of photoreceptor function determine the adequate therapeutic approach to use for each patient. Among such approaches, gene therapy is rapidly becoming a therapeutic reality applicable in the clinic. This massive move from laboratory work towards clinical application has been propelled by the advances in our understanding of disease genetics and mechanisms, gene delivery vectors, gene editing systems, and compensatory strategies for loss of photoreceptor function. Here, we provide an overview of existing modalities of retinal gene therapy and their relevance based on the needs of patient populations suffering from inherited retinal degenerations., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Long-term exposure to nicotine alters brain circuits and induces profound changes in decision-making strategies, affecting behaviors both related and unrelated to drug seeking and consumption. Using an intracranial self-stimulation reward-based foraging task, we investigated in mice the impact of chronic nicotine on midbrain dopamine neuron activity and its consequence on the trade-off between exploitation and exploration. Model-based and archetypal analysis revealed substantial inter-individual variability in decision-making strategies, with mice passively exposed to nicotine shifting toward a more exploitative profile compared to non-exposed animals. We then mimicked the effect of chronic nicotine on the tonic activity of dopamine neurons using optogenetics, and found that photo-stimulated mice adopted a behavioral phenotype similar to that of mice exposed to chronic nicotine. Our results reveal a key role of tonic midbrain dopamine in the exploration/exploitation trade-off and highlight a potential mechanism by which nicotine affects the exploration/exploitation balance and decision-making., (© 2021. The Author(s).)

The nucleoredoxin gene NXNL2 encodes for two products through alternative splicing, rod-derived cone viability factor-2 (RdCVF2) that mediates neuronal survival and the thioredoxin-related protein (RdCVF2L), an enzyme that regulates the phosphorylation of TAU. To investigate the link between NXNL2 and tauopathies, we studied the Nxnl2 knockout mouse (Nxnl2 -/- ). We established the expression pattern of the Nxnl2 gene in the brain using a Nxnl2 reporter mouse line, and characterized the behavior of the Nxnl2 -/- mouse at 2 months of age. Additionally, long term potentiation and metabolomic from hippocampal specimens were collected at 2 months of age. We studied TAU oligomerization, phosphorylation and aggregation in Nxnl2 -/- brain at 18 months of age. Finally, newborn Nxnl2 -/- mice were treated with adeno-associated viral vectors encoding for RdCVF2, RdCVF2L or both and measured the effect of this therapy on long-term potential, glucose metabolism and late-onset tauopathy. Nxnl2 -/- mice at 2 months of age showed severe behavioral deficiency in fear, pain sensitivity, coordination, learning and memory. The Nxnl2 -/- also showed deficits in long-term potentiation, demonstrating that the Nxnl2 gene is involved in regulating brain functions. Dual delivery of RdCVF2 and RdCVF2L in newborn Nxnl2 -/- mice fully correct long-term potentiation through their synergistic action. The expression pattern of the Nxnl2 gene in the brain shows a predominant expression in circumventricular organs, such as the area postrema. Glucose metabolism of the hippocampus of Nxnl2 -/- mice at 2 months of age was reduced, and was not corrected by gene therapy. At 18-month-old Nxnl2 -/- mice showed brain stigmas of tauopathy, such as oligomerization, phosphorylation and aggregation of TAU. This late-onset tauopathy can be prevented, albeit with modest efficacy, by recombinant AAVs administrated to newborn mice. The Nxnl2 -/- mice have memory dysfunction at 2-months that resembles mild-cognitive impairment and at 18-months exhibit tauopathy, resembling to the progression of Alzheimer's disease. We propose the Nxnl2 -/- mouse is a model to study multistage aged related neurodegenerative diseases. The NXNL2 metabolic and redox signaling is a new area of therapeutic research in neurodegenerative diseases., (Copyright © 2021 The Authors. Published by Elsevier B.V. All rights reserved.)