S., National Institutes of Health, USA; http://rsb.info.nih.gov/ij/) was used to first threshold and then select all Neurotrace-positive cells with a minimum pixel number of 175 reflecting a threshold diameter of ~25 m. were no clinical or histological signs of immune toxicity. Together these data indicate that delivery of AAV9 into the cisterna magna is an effective method for achieving gene transfer in the CNS, and suggest that adapting this uncommon injection method for human trials could vastly increase the efficiency of gene delivery. == Introduction == Gene therapy using AAV vectors offers enormous potential to treat obtained and congenital diseases affecting the central nervous system (CNS). Numerous studies in murine disease models have demonstrated the efficacy of AAV-mediated gene transfer in the brain, and initial human studies have shown an excellent safety profile. 15These pioneering clinical trials were carried out using multiple direct injections from the vector into the brain parenchyma, a strategy which has been shown in preclinical studies to achieve effective gene transfer to cells surrounding the injection site. 6However, treatment of diseases affecting cells throughout the CNS will ultimately necessitate a delivery strategy able of widespread transduction. Simply increasing the number of intraparenchymal injections is not feasible, as even the most promising large animal studies have required at least four injection sites, which would translate to more than 100 injections in an adult human. 6, 7Intravascular (IV) delivery of AAV serotype 9, which can traverse the blood-brain barrier, has been proposed as an alternative approach to achieve widespread transduction in the CNS. 8While IV delivery is extremely effective in mice, in larger animals transduction in the CNS has been much more restricted. 9, 10This approach is further limited by the extremely large doses required to achieve transduction in the brain and the resulting high off-target transduction of peripheral organs. 8, 9 Several groups have recently reported that delivery of AAV serotypes 7 and 9 into the cerebrospinal fluid (CSF) of nonhuman primates can effect transduction of both T-1095 neurons and glia throughout the brain and spinal cord. 1012This represents a critical advance in the field of CNS gene therapy. It may now be possible not only to achieve gene transfer throughout the CNS in humans, but to do so with a single minimally invasive injection. Access to the subarachnoid CSF is routinely achieved by lumbar puncture in clinical practice intended for diagnostic reasons and for supervision of medications. Although less common, access can also be achieved at the level of the cisterna magna by sub-occipital puncture. Intrathecal AAV delivery therefore represents a relatively low-risk delivery strategy that could be readily translated to the clinic. In order to advance this approach toward clinical trials, we performed nonhuman primate studies to evaluate the efficiency of two minimally invasive delivery approacheslumbar puncture T-1095 and suboccipital puncturefor achieving vector distribution throughout the brain and spinal cord. One previous study reported that both strategies result in transgene expression in the brain, but did not quantitatively compare vector distribution. 12Based on our findings we estimate that sub-occipital puncture is 10- to 100-fold more efficient intended for brain transduction, making this approach far more promising for clinical use. In addition , we performed detailed evaluation of histological markers of inflammation in the CNS of treated animals. T-1095 Several studies have found intrathecal delivery of AAV9 to be safe in nonhuman primates and other species, although one group offers reported that macaques treated with a vector carrying a GFP transgene experienced neurological deficits associated with histological evidence of an inflammatory response. 13We found that treated animals exhibited no clinical or histological abnormalities, although CSF lymphocytes were elevated in some animals. Overall, our data suggest that intrathecal AAV injection by suboccipital puncture could serve as a useful gene transfer method for the treatment of neurological disease. == Results == == Intracisternal delivery achieves broader vector distribution in the CNS == We treated six cynomolgus macaques with an intrathecal injection of a single-stranded AAV9 vector expressing GFP from either the cytomegalovirus (CMV) or chicken beta-actin (CB) promoter (Table 1). Four from the animals were injected via sub-occipital puncture; two were treated via lumbar puncture. There were no adverse clinical events during the course of the study. All animals were sacrificed 14 days after injection for histological analysis. == Table 1 . Summary of study subjects. == CM, cisterna magna; F, female; IV, intravenous; L, lumbar injection; ROA, route of Rabbit polyclonal to NOTCH1 administration. Vector distribution was evaluated in CNS and peripheral tissues by quantitative PCR (Figure 1). All animals treated by suboccipital puncture exhibited substantial vector deposition in the brain and spinal cord, with up to one vector genome per cell in most regions..