The most frequently used viral expression systems are developed from adenoviruses, adeno-associated virus (AAV), herpes simplex virus, gamma retroviruses and lentiviruses (LV; subclass of retroviruses), each having their particular advantages and limitations.1,19 AAV and LV are most often used in the central nervous system (CNS) since they are relatively easy to produce and evoke minimal immune responses.17,19 The packing capacity of AAV is low compared to LV, and genes delivered using AAV vectors do remain episomal in the transduced cell. Although LVs showed long-lasting expression2,20 and were clinically successful in several primate studies,21,22 AAV is considered to be more appealing for transgene expression in CNS. AAV is small (20 to 25 nm), nonenveloped, single-stranded DNA parvoviruses.23 They are known for their efficient neuronal (NeuN) transduction, long-lasting transgene expression (up to 2 years in rodents and 6 years in primates) and safety profile (no integration in host-cell genome).3,24–28 In addition, they can transduce both dividing and nondividing cells. The best-characterized AAV serotype, first cloned into bacterial plasmids, is AAV serotype 2 carrying an AAV2-derived genome (AAV2/2).29 Through cross-packaging, AAV2-based genomes can be packaged in alternative AAV serotypes (e.g., rAAV2/5), which yields higher transduction efficiencies than AAV2/2. AAV-based vectors are currently the most widely used for preclinical research, due to favorable characteristics such as higher transduction efficiencies and greater dispersion.30–32 Unfortunately, the latter properties are species specific and even brain region specific.30,33 Moreover, information about transduction and expression properties obtained in rodents does not always translate to primates.34–38 In macaque monkeys, a comparison between the transduction properties of different rAAVs in subcortical structures, including the substantia nigra, striatum,39,40 and basal ganglia,41 revealed that rAAV2/1 and rAAV2/5 were most efficient when under the control of a synthetic chicken ß-actin promoter. In addition, another study of Sanchez et al.40 revealed a three- to fourfold higher efficiency of rAAV2/8 compared to rAAV2/1, 2/2, and 2/5 when using a cytomegalovirus (CMV) promoter. Optogenetic studies in macaques confirmed that viral vector transduction in cortex is heterogenous across cortical layers.35,42,43 Recently, an exhaustive comparison of transduction efficiencies of various recombinant adeno-associated viral vector (rAAV) serotypes and promoters in marmoset, mouse, and macaque neocortex again revealed distinct transduction patterns depending on the serotype and promoter used in the viral vector.18 Serotype rAAV2 showed a smaller spread compared to rAAV1, 5, 8, and 9, while constructs with the SynI promoter showed higher transgene expression in layers 3 and 5 compared to CaMKII-constructs.