Heterogeneity of layer 4 in visual areas of rhesus macaque cortex.

Recently, single-cell RNA-sequencing (scRNA-seq) has enabled unprecedented insights to the cellular landscape of the brains of many different species, among them the rhesus macaque as a key animal model. Building on previous, broader surveys of the macaque brain, we closely examined five immediately neighboring areas within the visual cortex of the rhesus macaque: V1, V2, V4, MT and TEO. To facilitate this, we first devised a novel pipeline for brain spatial archive - the BrainSPACE - which enabled robust archiving and sampling from the whole unfixed brain. SnRNA-sequencing of ~100,000 nuclei from visual areas V1 and V4 revealed conservation within the GABAergic neuron subtypes, while seven and one distinct principle neuron subtypes were detected in V1 and V4, respectively, all most likely located in layer 4. Moreover, using small molecule fluorescence in situ hybridization, we identified cell type density gradients across V1, V2, V4, MT, and TEO appearing to reflect the visual hierarchy. These findings demonstrate an association between the clear areal specializations among neighboring areas with the hierarchical levels within the visual cortex of the rhesus macaque.

Lentiviral and adeno-associated vectors efficiently transduce mouse T lymphocytes when targeted to murine CD8.

Preclinical studies on gene delivery into mouse lymphocytes are often hampered by insufficient activity of lentiviral (LV) and adeno-associated vectors (AAVs) as well as missing tools for cell type selectivity when considering in vivo gene therapy. Here, we selected designed ankyrin repeat proteins (DARPins) binding to murine CD8. The top-performing DARPin was displayed as targeting ligand on both vector systems. When used on engineered measles virus (MV) glycoproteins, the resulting mCD8-LV transduced CD8+ mouse lymphocytes with near-absolute (>99%) selectivity. Despite its lower functional titer, mCD8-LV achieved 4-fold higher gene delivery to CD8+ cells than conventional VSV-LV when added to whole mouse blood. Addition of mCD8-LV encoding a chimeric antigen receptor (CAR) specific for mouse CD19 to splenocytes resulted in elimination of B lymphocytes and lymphoma cells. For display on AAV, the DARPin was inserted into the GH2-GH3 loop of the AAV2 capsid protein VP1, resulting in a DARPin-targeted AAV we termed DART-AAV. Stocks of mCD8-AAV contained similar genome copies as AAV2 but were >20-fold more active in gene delivery in mouse splenocytes, while exhibiting >99% specificity for CD8+ cells. These results suggest that receptor targeting can overcome blocks in transduction of mouse splenocytes.

Optimization of production and transgene expression of a retrogradely transported pseudotyped lentiviral vector.

BACKGROUND: To target specific neuronal populations by gene transfer is challenging. A complicating fact is that populations of neurons may have opposing roles despite being found adjacent to each other. One example is the medium spiny neurons of the striatum. These cells have different projection patterns, a trait used in this study to specifically target one population. NEW METHOD: Here we present a way of labeling and further studying neurons based on their projections. This was achieved by pseudotyping lentiviral vectors with a chimeric glycoprotein allowing for retrograde transport in combination with optimizing the promoter element used. RESULTS: We transduced on average 4000 neurons of the direct pathway in the striatum, with the viral vector allowing for microscopy and miRNA immunoprecipitation. In addition, we were able to optimize vector production, reducing the time and material used. COMPARISON WITH EXISTING METHOD: The optimized protocol is more reproducible compared to previously published protocols. Alternative methods to study specific populations of neurons are transgenic animals or, if available, specific promoter elements. However, very specific promoter elements are rarely available and often large, limiting the usefulness in viral vectors. Our optimized retrograde vectors allow for selection based on neuronal projections and are therefore independent of such elements. CONCLUSION: We have developed a method that allows for specific analysis of neuronal subpopulations in the brain either by microscopy or by biochemical methods e.g. immunoprecipitation. This method is simple to use and can be combined with transgenic animals for studying disease models.