In this report, we describe the development of a modified adeno-associated virus (AAV) capsid and promoter for transduction of retinal ON-bipolar cells. The bipolar cells, which are post-synaptic to the photoreceptors, are important retinal targets for both basic and preclinical research. In particular, a therapeutic strategy under investigation for advanced forms of blindness involves using optogenetic molecules to render ON-bipolar cells light-sensitive. Currently, delivery of adequate levels of gene expression is a limiting step for this approach. The synthetic AAV capsid and promoter described here achieves high level of optogenetic transgene expression in ON-bipolar cells. This evokes high-frequency (∼100 Hz) spiking responses in ganglion cells of previously blind, rd1, mice. Our vector is a promising vehicle for further development toward potential clinical use.

Targeting genes to specific neuronal or glial cell types is valuable both for understanding and for repairing brain circuits. Adeno-associated viral vectors (AAVs) are frequently used for gene delivery, but targeting expression to specific cell types is a challenge. We created a library of 230 AAVs, each with a different synthetic promoter designed using four independent strategies. We show that ~11% of these AAVs specifically target expression to neuronal and glial cell types in the mouse retina, mouse brain, non-human primate retina in vivo, and in the human retina in vitro. We demonstrate applications for recording, stimulation, and molecular characterization, as well as the intersectional and combinatorial labeling of cell types. These resources and approaches allow economic, fast, and efficient cell-type targeting in a variety of species, both for fundamental science and for gene therapy.Despite the central importance for both basic and translational research, most current technologies available for cell-type-targeting rely on transgenic animals, which limits their applicability. Either the genetic tool that senses or modulates brain function, or the enzyme, such as Cre recombinase, that allows the genetic tool to be conditionally expressed, is expressed from the animal's genome. The inclusion of a transgenic component in the cell-type-targeting strategy excludes its use in therapy for humans, limits its range of application in pre-clinical, non-human primate research, and complicates its use in model organisms such as mice. The development of transgenic non-human primates and mice is costly and slow, especially since cell-type targeting is often applied in the context of other genetic manipulations, such as double or triple gene knockouts, or when targeting different cell types with different tools.Viral vectors for cell-type-targeting may overcome such limitations. AAVs are the most frequently used vectors in both basic research and gene therapy, as they are safe for use in all tested species, including humans and non-human primates, and their production is simple, cheap, and fast (Planul and Dalkara, 2017). They have three important components: the capsid for cell entry, the promoter that drives transgene expression, and the gene of interest to be expressed in the transduced cells, and they drive expression episomally (Duan et al., 1998; Penaud-Budloo et al., 2008). Futhermore, many genetic tools are small enough to fit into AAVs, different AAVs can be injected together, and synthetic AAV capsids allow brain-wide delivery (Deverman et al., 2016).Cell-type-targeting by AAVs could be achieved by engineering the capsid and/or by using specific promoters. Capsid protein mutations can be used to tune the efficacy of

The comparative study of cell types is a powerful approach toward deciphering animal evolution. To avoid selection biases, however, comparisons ideally involve all cell types present in a multicellular organism. Here, we use image registration and a newly developed "Profiling by Signal Probability Mapping" algorithm to generate a cellular resolution 3D expression atlas for an entire animal. We investigate three-segmented young worms of the marine annelid Platynereis dumerilii, with a rich diversity of differentiated cells present in relatively low number. Starting from whole-mount expression images for close to 100 neural specification and differentiation genes, our atlas identifies and molecularly characterizes 605 bilateral pairs of neurons at specific locations in the ventral nerve cord. Among these pairs, we identify sets of neurons expressing similar combinations of transcription factors, located at spatially coherent anterior-posterior, dorsal-ventral, and mediallateral coordinates that we interpret as cell types. Comparison with motor and interneuron types in the vertebrate neural tube indicates conserved combinations, for example, of cell types cospecified by Gata1/2/3 and Tal transcription factors. These include V2b interneurons and the central spinal fluid-contacting Kolmer-Agduhr cells in the vertebrates, and several neuron types in the intermediate ventral ganglionic mass in the annelid. We propose that Kolmer-Agduhr cell-like mechanosensory neurons formed part of the mucociliary sole in protostome-deuterostome ancestors and diversified independently into several neuron types in annelid and vertebrate descendants.gene-expression atlas | evo-devo | cell-type evolution | Kolmer-Agduhr cells | ProSPr