Gene therapy using recombinant adeno‐associated virus (rAAV) vectors to treat blinding retinal dystrophies has become clinical reality. Therapeutically impactful targeting of photoreceptors still relies on subretinal vector delivery, which detaches the retina and harbours substantial risks of collateral damage, often without achieving widespread photoreceptor transduction. Herein, we report the development of novel engineered rAAV vectors that enable efficient targeting of photoreceptors via less invasive intravitreal administration. A unique in vivo selection procedure was performed, where an AAV2‐based peptide‐display library was intravenously administered in mice, followed by isolation of vector DNA from target cells after only 24 h. This stringent selection yielded novel vectors, termed AAV2.GL and AAV2.NN, which mediate widespread and high‐level retinal transduction after intravitreal injection in mice, dogs and non‐human primates. Importantly, both vectors efficiently transduce photoreceptors in human retinal explant cultures. As proof‐of‐concept, intravitreal Cnga3 delivery using AAV2.GL lead to cone‐specific expression of Cnga3 protein and rescued photopic cone responses in the Cnga3−/− mouse model of achromatopsia. These novel rAAV vectors expand the clinical applicability of gene therapy for blinding human retinal dystrophies.

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Many neurodegenerative diseases cause degeneration of specific types of neurons. For example, glaucoma leads to death of retinal ganglion cells, leaving other neurons intact. Neurons are not regenerated in the adult mammalian central nervous system. However, in nonmammalian vertebrates, glial cells spontaneously reprogram into neural progenitors and replace neurons after injury. We have recently developed strategies to stimulate regeneration of functional neurons in the adult mouse retina by overexpressing the proneural factor Ascl1 in Müller glia. Here, we test additional transcription factors (TFs) for their ability to direct regeneration to particular types of retinal neurons. We engineered mice to express different combinations of TFs in Müller glia, including Ascl1, Pou4f2, Islet1, and Atoh1. Using immunohistochemistry, single-cell RNA sequencing, single-cell assay for transposase-accessible chromatin sequencing, and electrophysiology, we find that retinal ganglion–like cells can be regenerated in the damaged adult mouse retina in vivo with targeted overexpression of developmental retinal ganglion cell TFs.

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