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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.