AAV PHP.eB-CAG-GFP

The development of highly effective central nervous system (CNS)-targeted AAV gene therapies is critical to address challenges in preclinical and clinical research. The engineered capsids AAV.PHP.B and AAV.PHP.eB showed significantly improved blood-brain barrier penetration compared with the parental serotype AAV9, but the effect varied by animal system, strain, and delivery route. Here, researchers evaluated the comparative transduction efficiencies of equititer doses (6 × 1011vg) of AAV.PHP.eB-CAG-GFP and AAV9-CAG-GFP when delivered into the cisterna magna of 6–9-month-old rats. Through quantitative and qualitative assessments, the researchers observed that the biodistribution of GFP+ cells and fibers was consistently better in animals treated with AAV.PHP.eB compared to animals treated with AAV9. In animals treated with AAV.PHP.eB, the GFP signal was uniformly enhanced throughout the craniocaudal brain region, and matching GFP protein expression was detected in the forebrain, midbrain, and cerebellum. Taken together, these data illustrate the benefit of intracisternal infusions of AAV.PHP.eB as an optimal system to distribute CNS gene therapies in preclinical investigations of rats, and may have important translational implications for the clinical CNS targeting.

Here, researchers report the results of a rapid, simplified intrathecal injection protocol. Middle-aged rats were mounted into a stereotaxic frame and oriented to elevate the occipital ridge and facilitate access to the cisternal membrane. 50 µL of saline, AAV.PHP.eB-CAG-GFP (AAV.PHP.eB-GFP), or AAV9-CAG-GFP (AAV9-GFP) was injected into the cisterna magna, and animals were sacrificed 1 month after injection(Figure 1a). The relative transduction profiles of AAV.PHP.eB-GFP and AAV9-GFP following equal, high-dose ICM injection were first assessed qualitatively. Rats injected with AAV.PHP.eB-GFP had significantly higher GFP expression throughout the rostral and caudal range of the brain (Figure 1b). Transduction was most striking in the clusters of GFP+ perinuclear cell patterns observed throughout the cortex, primarily spanning cortical layers III-V in AAV.PHP.eB-GFP treated animals (Figure 1b, c). In contrast, AAV9-GFP treated animals had sparse staining, with minimal fiber staining throughout the body and greater GFP expression in the cerebellum and CM proximal nuclei (Figure 1b,c). AAV.PHP.eB-treated animals showed enhanced cellular staining in periventricular areas such as septal nuclei or ventral hippocampus (Figure 1c). However, enhanced GFP+ expression was not restricted to the periventricular region, implying that viral transduction efficiency is not solely directly related to CSF contact (Figure 1c). Cerebral and cerebellar cortical GFP expression is more prominent throughout AAV.PHP.eB-treated subjects, whereas subcortical regions (striatum/thalamus) feature high levels of GFP + puncta compared to that of AAV9-treated subjects (Figure 1c). Increased AAV.PHP.eB-GFP expression was also observed in white and gray matter regions along the lateral funiculus of the cervical spinal cord (Figure 1c).

Figure 1. Expansive CNS transduction via intracisternal delivery of AAV.PHP.eB-CAG-GFP. (Chatterjee D, et al., 2022)