GDNF gene therapy for alcohol use disorder in male non-human primates.

Alcohol use disorder (AUD) exacts enormous personal, social and economic costs globally. Return to alcohol use in treatment-seeking patients with AUD is common, engendered by a cycle of repeated abstinence-relapse episodes even with use of currently available pharmacotherapies. Repeated ethanol use induces dopaminergic signaling neuroadaptations in ventral tegmental area (VTA) neurons of the mesolimbic reward pathway, and sustained dysfunction of reward circuitry is associated with return to drinking behavior. We tested this hypothesis by infusing adeno-associated virus serotype 2 vector encoding human glial-derived neurotrophic factor (AAV2-hGDNF), a growth factor that enhances dopaminergic neuron function, into the VTA of four male rhesus monkeys, with another four receiving vehicle, following induction of chronic alcohol drinking. GDNF expression ablated the return to alcohol drinking behavior over a 12-month period of repeated abstinence-alcohol reintroduction challenges. This behavioral change was accompanied by neurophysiological modulations to dopamine signaling in the nucleus accumbens that countered the hypodopaminergic signaling state associated with chronic alcohol use, indicative of a therapeutic modulation of limbic circuits countering the effects of alcohol. These preclinical findings suggest gene therapy targeting relapse prevention may be a potential therapeutic strategy for AUD.

Targeted interleukin-10 plasmid DNA therapy in the treatment of osteoarthritis: Toxicology and pain efficacy assessments.

Osteoarthritis results in chronic pain and loss of function. Proinflammatory cytokines create both osteoarthritis pathology and pain. Current treatments are poorly effective, have significant side effects, and have not targeted the cytokines central to osteoarthritis development and maintenance. Interleukin-10 is an anti-inflammatory cytokine that potently and broadly suppresses proinflammatory cytokine activity. However, interleukin-10 protein has a short half-life in vivo and poor joint permeability. For sustained IL-10 activity, we developed a plasmid DNA-based therapy that expresses a long-acting human interleukin-10 variant (hIL-10var). Here, we describe the 6-month GLP toxicology study of this therapy. Intra-articular injections of hIL-10var pDNA into canine stifle joints up to 1.5 mg bilaterally were well-tolerated and without pathologic findings. This represents the first long-term toxicologic assessment of intra-articular pDNA therapy. We also report results of a small double-blind, placebo-controlled study of the effect of intra-articular hIL-10var pDNA on pain measures in companion (pet) dogs with naturally occurring osteoarthritis. This human IL-10-based targeted therapy reduced pain measures in the dogs, based on veterinary and owner ratings, without any adverse findings. These results with hIL-10var pDNA therapy, well-tolerated and without toxicologic effects, establish the basis for clinical trials of a new class of safe and effective therapies for OA.

Adeno-associated viral vector serotype 9-based gene therapy for Niemann-Pick disease type A.

Niemann-Pick disease type A (NPD-A) is a lysosomal storage disorder characterized by neurodegeneration and early death. It is caused by loss-of-function mutations in the gene encoding for acid sphingomyelinase (ASM), which hydrolyzes sphingomyelin into ceramide. Here, we evaluated the safety of cerebellomedullary (CM) cistern injection of adeno-associated viral vector serotype 9 encoding human ASM (AAV9-hASM) in nonhuman primates (NHP). We also evaluated its therapeutic benefit in a mouse model of the disease (ASM-KO mice). We found that CM injection in NHP resulted in widespread transgene expression within brain and spinal cord cells without signs of toxicity. CM injection in the ASM-KO mouse model resulted in hASM expression in cerebrospinal fluid and in different brain areas without triggering an inflammatory response. In contrast, direct cerebellar injection of AAV9-hASM triggered immune response. We also identified a minimally effective therapeutic dose for CM injection of AAV9-hASM in mice. Two months after administration, the treatment prevented motor and memory impairment, sphingomyelin (SM) accumulation, lysosomal enlargement, and neuronal death in ASM-KO mice. ASM activity was also detected in plasma from AAV9-hASM CM-injected ASM-KO mice, along with reduced SM amount and decreased inflammation in the liver. Our results support CM injection for future AAV9-based clinical trials in NPD-A as well as other lysosomal storage brain disorders.

Kinetics and MR-Based Monitoring of AAV9 Vector Delivery into Cerebrospinal Fluid of Nonhuman Primates.

Here we evaluated the utility of MRI to monitor intrathecal infusions in nonhuman primates. Adeno-associated virus (AAV) spiked with gadoteridol, a gadolinium-based MRI contrast agent, enabled real-time visualization of infusions delivered either via cerebromedullary cistern, lumbar, cerebromedullary and lumbar, or intracerebroventricular infusion. The kinetics of vector clearance from the cerebrospinal fluid (CSF) were analyzed. Our results highlight the value of MRI in optimizing the delivery of infusate into CSF. In particular, MRI revealed differential patterns of infusate distribution depending on the route of delivery. Gadoteridol coverage analysis showed that cerebellomedullary cistern delivery was a reliable and effective route of injection, achieving broad infusate distribution in the brain and spinal cord, and was even greater when combined with lumbar injection. In contrast, intracerebroventricular injection resulted in strong cortical coverage but little spinal distribution. Lumbar injection alone led to the distribution of MRI contrast agent mainly in the spinal cord with little cortical coverage, but this delivery route was unreliable. Similarly, vector clearance analysis showed differences between different routes of delivery. Overall, our data support the value of monitoring CSF injections to dissect different patterns of gadoteridol distribution based on the route of intrathecal administration.

Extensive Transduction and Enhanced Spread of a Modified AAV2 Capsid in the Non-human Primate CNS.

The present study was designed to characterize transduction of non-human primate brain and spinal cord with a modified adeno-associated virus serotype 2, incapable of binding to the heparan sulfate proteoglycan receptor, referred to as AAV2-HBKO. AAV2-HBKO was infused into the thalamus, intracerebroventricularly or via a combination of both intracerebroventricular and thalamic delivery. Thalamic injection of this modified vector encoding GFP resulted in widespread CNS transduction that included neurons in deep cortical layers, deep cerebellar nuclei, several subcortical regions, and motor neuron transduction in the spinal cord indicative of robust bidirectional axonal transport. Intracerebroventricular delivery similarly resulted in widespread cortical transduction, with one striking distinction that oligodendrocytes within superficial layers of the cortex were the primary cell type transduced. Robust motor neuron transduction was also observed in all levels of the spinal cord. The combination of thalamic and intracerebroventricular delivery resulted in transduction of oligodendrocytes in superficial cortical layers and neurons in deeper cortical layers. Several subcortical regions were also transduced. Our data demonstrate that AAV2-HBKO is a powerful vector for the potential treatment of a wide number of neurological disorders, and highlight that delivery route can significantly impact cellular tropism and pattern of CNS transduction.

Ganglioside Metabolism and Parkinson's Disease.

Here we advance the hypothesis that Parkinson's disease (PD) is fundamentally a failure of trophic support for specific classes of neurons, primarily catecholaminergic. Evidence from our laboratory provides a framework into which a broad array of findings from many quarters can be integrated into a general theory that offers testable hypotheses to new and established investigators. Mice deficient in the ability to synthesize series-a gangliosides, specifically GM1 ganglioside, develop parkinsonism. We found that this seems to be due to a failure in signaling efficiency by the important catecholaminergic growth factor, GDNF. Interestingly, these mice accumulate alpha-synuclein in nigral neurons. Striatal over-expression of GDNF eliminates these aggregates and also restores normal motor function. These findings bring into question common beliefs about alpha-synuclein pathology and may help us to reinterpret other experimental findings in a new light. The purpose of this article is to provoke new thinking about PD and hopefully encourage younger scientists to explore some of the ideas presented below.

Soluble FLT1 Gene Therapy Alleviates Brain Arteriovenous Malformation Severity.

BACKGROUND AND PURPOSE: Brain arteriovenous malformation (bAVM) is an important risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. Excessive vascular endothelial growth factor has been implicated in bAVM pathophysiology. Because soluble FLT1 binds to vascular endothelial growth factor with high affinity, we tested intravenous delivery of an adeno-associated viral vector serotype-9 expressing soluble FLT1 (AAV9-sFLT1) to alleviate the bAVM phenotype. METHODS: Two mouse models were used. In model 1, bAVM was induced in R26CreER;Eng2f/2f mice through global Eng gene deletion and brain focal angiogenic stimulation; AAV2-sFLT02 (an AAV expressing a shorter form of sFLT1) was injected into the brain at the time of model induction, and AAV9-sFLT1, intravenously injected 8 weeks after. In model 2, SM22αCre;Eng2f/2f mice had a 90% occurrence of spontaneous bAVM at 5 weeks of age and 50% mortality at 6 weeks; AAV9-sFLT1 was intravenously delivered into 4- to 5-week-old mice. Tissue samples were collected 4 weeks after AAV9-sFLT1 delivery. RESULTS: AAV2-sFLT02 inhibited bAVM formation, and AAV9-sFLT1 reduced abnormal vessels in model 1 (GFP versus sFLT1: 3.66±1.58/200 vessels versus 1.98±1.29, P<0.05). AAV9-sFLT1 reduced the occurrence of bAVM (GFP versus sFLT1: 100% versus 36%) and mortality (GFP versus sFLT1: 57% [12/22 mice] versus 24% [4/19 mice], P<0.05) in model 2. Kidney and liver function did not change significantly. Minor liver inflammation was found in 56% of AAV9-sFLT1-treated model 1 mice. CONCLUSIONS: By applying a regulated mechanism to restrict sFLT1 expression to bAVM, AAV9-sFLT1 can potentially be developed into a safer therapy to reduce the bAVM severity.

Depletion of AADC activity in caudate nucleus and putamen of Parkinson's disease patients; implications for ongoing AAV2-AADC gene therapy trial.

In Parkinson's disease (PD), aromatic L-amino acid decarboxylase (AADC) is the rate-limiting enzyme in the conversion of L-DOPA (Sinemet) to dopamine (DA). Previous studies in PD animal models demonstrated that lesion of dopaminergic neurons is associated with profound loss of AADC activity in the striatum, blocking efficient conversion of L-DOPA to DA. Relatively few studies have directly analyzed AADC in PD brains. Thus, the aim of this study was to gain a better understanding of regional changes in AADC activity, DA, serotonin and their monoamine metabolites in the striatum of PD patients and experimentally lesioned animals (rat and MPTP-treated nonhuman primate, NHP). Striatal AADC activity was determined post mortem in neuropathologically confirmed PD subjects, animal models and controls. A regional analysis was performed for striatal AADC activity and monoamine levels in NHP tissue. Interestingly, analysis of putaminal AADC activity revealed that control human striatum contained much less AADC activity than rat and NHP striata. Moreover, a dramatic loss of AADC activity in PD striatum compared to controls was detected. In MPTP-treated NHP, caudate nucleus was almost as greatly affected as putamen, although mean DA turnover was higher in caudate nucleus. Similarly, DA and DA metabolites were dramatically reduced in different regions of PD brains, including caudate nucleus, whereas serotonin was relatively spared. After L-DOPA administration in MPTP-treated NHP, very poor conversion to DA was detected, suggesting that AADC in NHP nigrostriatal fibers is mainly responsible for L-DOPA to DA conversion. These data support further the rationale behind viral gene therapy with AAV2-hAADC to restore AADC levels in putamen and suggest further the advisability of expanding vector delivery to include coverage of anterior putamen and the caudate nucleus.

Behavioral assessment of neuropathic pain, fatigue, and anxiety in experimental autoimmune encephalomyelitis (EAE) and attenuation by interleukin-10 gene therapy.

Relapsing-remitting multiple sclerosis is commonly associated with motor impairments, neuropathic pain, fatigue, mood disorders, and decreased life expectancy. However, preclinical pharmacological studies predominantly rely on clinical scoring of motor deficit as the sole behavioral endpoint. Thus, the translational potential of these studies is limited. Here, we have assessed the therapeutic potential of a novel anti-inflammatory interleukin-10 (IL-10) non-viral gene therapy formulation (XT-101-R) in a rat relapsing remitting experimental autoimmune encephalomyelitis (EAE) model. EAE induced motor deficits and neuropathic pain as reflected by induction of low-threshold mechanical allodynia, suppressed voluntary wheel running, decreased social exploration, and was associated with markedly enhanced mortality. We also noted that voluntary wheel running was depressed prior to the onset of motor deficit, and may therefore serve as a predictor of clinical symptoms onset. XT-101-R was intrathecally dosed only once at the onset of motor deficits, and attenuated each of the EAE-induced symptoms and improved survival, relative to vehicle control. This is the first pharmacological assessment of such a broad range of EAE symptoms, and provides support for IL-10 gene therapy as a clinical strategy for the treatment of multiple sclerosis.

Widespread AAV1- and AAV2-mediated transgene expression in the nonhuman primate brain: implications for Huntington's disease.

Huntington's disease (HD) is caused by a toxic gain-of-function associated with the expression of the mutant huntingtin (htt) protein. Therefore, the use of RNA interference to inhibit Htt expression could represent a disease-modifying therapy. The potential of two recombinant adeno-associated viral vectors (AAV), AAV1 and AAV2, to transduce the cortico-striatal tissues that are predominantly affected in HD was explored. Green fluorescent protein was used as a reporter in each vector to show that both serotypes were broadly distributed in medium spiny neurons in the striatum and cortico-striatal neurons after infusion into the putamen and caudate nucleus of nonhuman primates (NHP), with AAV1-directed expression being slightly more robust than AAV2-driven expression. This study suggests that both serotypes are capable of targeting neurons that degenerate in HD, and it sets the stage for the advanced preclinical evaluation of an RNAi-based therapy for this disease.

AAV viral vector delivery to the brain by shape-conforming MR-guided infusions.

Gene transfer technology offers great promise as a potential therapeutic approach to the brain but has to be viewed as a very complex technology. Success of ongoing clinical gene therapy trials depends on many factors such as selection of the correct genetic and anatomical target in the brain. In addition, selection of the viral vector capable of transfer of therapeutic gene into target cells, along with long-term expression that avoids immunotoxicity has to be established. As with any drug development strategy, delivery of gene therapy has to be consistent and predictable in each study subject. Failed drug and vector delivery will lead to failed clinical trials. In this article, we describe our experience with AAV viral vector delivery system, that allows us to optimize and monitor in real time viral vector administration into affected regions of the brain. In addition to discussing MRI-guided technology for administration of AAV vectors we have developed and now employ in current clinical trials, we also describe ways in which infusion cannula design and stereotactic trajectory may be used to maximize the anatomical coverage by using fluid backflow. This innovative approach enables more precise coverage by fitting the shape of the infusion to the shape of the anatomical target.

Cerebellomedullary Cistern Delivery for AAV-Based Gene Therapy: A Technical Note for Nonhuman Primates.

Accessing cerebrospinal fluid (CSF) from the craniocervical junction through the posterior atlanto-occipital membrane via cerebellomedullary injection (also known as cisternal puncture or cisterna magna injection) has become a standard procedure in preclinical studies. Such delivery provides broader coverage to the central and peripheral nervous system unlike local parenchymal delivery alone. As a clinical application, this approach offers a more reliable method for neurological gene replacement delivery in infants, where skull-mounted devices are not indicated. Here we describe a consistent, precise, and safe method for CSF injection with minimal equipment and technical skills.

Peri-tumoral leakage during intra-tumoral convection-enhanced delivery has implications for efficacy of peri-tumoral infusion before removal of tumor.

In cases of malignant brain tumors, infiltrating tumor cells that exist at the tumor-surrounding brain tissue always escape from cytoreductive surgery and, protected by blood-brain barrier (BBB), survive the adjuvant chemoradiotherapy, eventually leading to tumor recurrence. Local interstitial delivery of chemotherapeutic agents is a promising strategy to target these cells. During our effort to develop effective drug delivery methods by intra-tumoral infusion of chemotherapeutic agents, we found consistent pattern of leakage from the tumor. Here we describe our findings and propose promising strategy to cover the brain tissue surrounding the tumor with therapeutic agents by means of convection-enhanced delivery. First, the intracranial tumor isograft model was used to define patterns of leakage from tumor mass after intra-tumoral infusion of the chemotherapeutic agents. Liposomal doxorubicin, although first distributed inside the tumor, distributed diffusely into the surrounding normal brain once the leakage happen. Trypan blue dye was used to evaluate the distribution pattern of peri-tumoral infusions. When infused intra- or peri-tumorally, infusates distributed robustly into the tumor border. Subsequently, volume of distributions with different infusion scheduling; including intra-tumoral infusion, peri-tumoral infusion after tumor resection, peri-tumoral infusion without tumor removal with or without systemic infusion of steroids, were compared with Evans-blue dye. Peri-tumoral infusion without tumor removal resulted in maximum volume of distribution. Prior use of steroids further increased the volume of distribution. Local interstitial drug delivery targeting tumor surrounding brain tissue before tumor removal should be more effective when targeting the invading cells.

Self-assembled 20-nm (64)Cu-micelles enhance accumulation in rat glioblastoma.

There is an urgent need to develop nanocarriers for the treatment of glioblastoma multiforme (GBM). Using co-registered positron emission tomography (PET) and magnetic resonance (MR) images, here we performed systematic studies to investigate how a nanocarrier's size affects the pharmacokinetics and biodistribution in rodents with a GBM xenograft. In particular, highly stable, long-circulating three-helix micelles (3HM), based on a coiled-coil protein tertiary structure, were evaluated as an alternative to larger nanocarriers. While the circulation half-life of the 3HM was similar to 110-nm PEGylated liposomes (t1/2=15.5 and 16.5h, respectively), the 20-nm micelles greatly enhanced accumulation within a U87MG xenograft in nu/nu rats after intravenous injection. After accounting for tumor blood volume, the extravasated nanoparticles were quantified from the PET images, yielding ~0.77%ID/cm(3) for the micelles and 0.45%ID/cm(3) for the liposomes. For GBM lesions with a volume greater than 100mm(3), 3HM accumulation was enhanced both within the detectable tumor and in the surrounding brain parenchyma. Further, the nanoparticle accumulation was shown to extend to the margins of the GBM xenograft. In summary, 3HM provides an attractive nanovehicle for carrying treatment to GBM.

Carbidopa-based modulation of the functional effect of the AAV2-hAADC gene therapy in 6-OHDA lesioned rats.

Progressively blunted response to L-DOPA in Parkinson's disease (PD) is a critical factor that complicates long-term pharmacotherapy in view of the central importance of this drug in management of the PD-related motor disturbance. This phenomenon is likely due to progressive loss of one of the key enzymes involved in the biosynthetic pathway for dopamine in the basal ganglia: aromatic L-amino acid decarboxylase (AADC). We have developed a gene therapy based on an adeno-associated virus encoding human AADC (AAV2-hAADC) infused into the Parkinsonian striatum. Although no adverse clinical effects of the AAV2-hAADC gene therapy have been observed so far, the ability to more precisely regulate transgene expression or transgene product activity could be an important long-term safety feature. The present study was designed to define pharmacological regulation of the functional activity of AAV2-hAADC transgene product by manipulating L-DOPA and carbidopa (AADC inhibitor) administration in hemi-parkinsonian rats. Thirty days after unilateral striatal infusion of AAV2-hAADC, animals displayed circling behavior and acceleration of dopamine metabolism in the lesioned striatum after administration of a low dose of L-DOPA (5 mg/kg) co-administered with 1.25 mg/kg of carbidopa. This phenomenon was not observed in control AAV2-GFP-treated rats. Withdrawal of carbidopa from a daily L-DOPA regimen decreased the peripheral L-DOPA pool, resulting in almost total loss of L-DOPA-induced behavioral response in AAV2-hAADC rats and a significant decline in striatal dopamine turnover. The serum L-DOPA level correlated with the magnitude of circling behavior in AAV2-hAADC rats. Additionally, AADC activity in homogenates of lesioned striata transduced by AAV2-AADC was 10-fold higher when compared with AAV2-GFP-treated control striata, confirming functional transduction. Our data suggests that the pharmacological regulation of circulating L-DOPA might be effective in the controlling of function of AAV2-hAADC transgene product in PD gene therapy.

GDNF signaling implemented by GM1 ganglioside; failure in Parkinson's disease and GM1-deficient murine model.

GDNF is indispensible for adult catecholaminergic neuron survival, and failure of GDNF signaling has been linked to loss of dopaminergic neurons in Parkinson's disease (PD). This study demonstrates attenuated GDNF signaling in neurons deficient in ganglio-series gangliosides, and restoration of such signaling with LIGA20, a membrane permeable analog of GM1. GM1 is shown to associate in situ with GFRα1 and RET, the protein components of the GDNF receptor, this being necessary for assembly of the tripartite receptor complex. Mice wholly or partially deficient in GM1 due to disruption of the B4galnt1 gene developed PD symptoms based on behavioral and neuropathological criteria which were largely ameliorated by gene therapy with AAV2-GDNF and also with LIGA20 treatment. The nigral neurons of PD subjects that were severely deficient in GM1 showed subnormal levels of tyrosine phosphorylated RET. Also in PD brain, GM1 levels in the occipital cortex, a region of limited PD pathology, were significantly below age-matched controls, suggesting the possibility of systemic GM1 deficiency as a risk factor in PD. This would accord with our finding that mice with partial GM1 deficiency represent a faithful recapitulation of the human disease. Together with the previously demonstrated age-related decline of GM1 in human brain, this points to gradual development of subthreshold levels of GM1 in the brain of PD subjects below that required for effective GDNF signaling. This hypothesis offers a dramatically different explanation for the etiology of sporadic PD as a manifestation of acquired resistance to GDNF.

Glial cell line-derived neurotrophic factor (GDNF) is an endogenous protector in the mesolimbic system against excessive alcohol consumption and relapse.

Moderate social consumption of alcohol is common; however, only a small percentage of individuals transit from social to excessive, uncontrolled alcohol drinking. This suggests the existence of protective mechanisms that prevent the development of alcohol addiction. Here, we tested the hypothesis that the glial cell line-derived neurotrophic factor (GDNF) in the mesolimbic system [e.g. the nucleus accumbens (Acb) and ventral tegmental area (VTA)] is part of such a mechanism. We found that GDNF knockdown, by infecting rat Acb neurons with a small hairpin RNA (shRNA) targeting the GDNF gene, produced a rapid escalation to excessive alcohol consumption and enhanced relapse to alcohol drinking. Conversely, viral-mediated overexpression of the growth factor in the mesolimbic system blocked the escalation from moderate to excessive alcohol drinking. To access the mechanism underlying GDNF's actions, we measured the firing rate of dopaminergic (DAergic) neurons in the VTA after a history of excessive alcohol intake with or without elevating GDNF levels. We found that the spontaneous firing rate of DAergic neurons in the VTA was reduced during alcohol withdrawal and that GDNF reversed this alcohol-induced DA deficiency. Together, our results suggest that endogenous GDNF in the mesolimbic system controls the transition from moderate to excessive alcohol drinking and relapse via reversal of alcohol-dependent neuro-adaptations in DAergic VTA neurons.

SAFETY AND TOLERABILITY OF MRI-GUIDED INFUSION OF AAV2-hAADC INTO THE MID-BRAIN OF NON-HUMAN PRIMATE.

Aromatic L-amino acid decarboxylase (AADC) deficiency is a rare, autosomal-recessive neurological disorder caused by mutations in the DDC gene that leads to an inability to synthesize catecholamines and serotonin. As a result, patients suffer compromised development, particularly in motor function. A recent gene replacement clinical trial explored putaminal delivery of recombinant adeno-associated virus serotype 2 vector encoding human AADC (AAV2-hAADC) in AADC-deficient children. Unfortunately, patients presented only modest amelioration of motor symptoms, which authors acknowledged could be due to insufficient transduction of putamen. We hypothesize that, with the development of a highly accurate MRI-guided cannula placement technology, a more effective approach might be to target the affected mid-brain neurons directly. Transduction of AADC-deficient dopaminergic neurons in the substantia nigra and ventral tegmental area with locally infused AAV2-hAADC would be expected to lead to restoration of normal dopamine levels in affected children. The objective of this study was to assess the long-term safety and tolerability of bilateral AAV2-hAADC MRI-guided pressurized infusion into the mid-brain of non-human primates. Animals received either vehicle, low or high AAV2-hAADC vector dose and were euthanized 1, 3 or 9 months after surgery. Our data indicate that effective mid-brain transduction was achieved without untoward effects.

Distribution of nanoparticles throughout the cerebral cortex of rodents and non-human primates: Implications for gene and drug therapy.

When nanoparticles/proteins are infused into the brain, they are often transported to distal sites in a manner that is dependent both on the characteristics of the infusate and the region targeted. We have previously shown that adeno-associated virus (AAV) is disseminated within the brain by perivascular flow and also by axonal transport. Perivascular distribution usually does not depend strongly on the nature of the infusate. Many proteins, neutral liposomes and AAV particles distribute equally well by this route when infused under pressure into various parenchymal locations. In contrast, axonal transport requires receptor-mediated uptake of AAV by neurons and engagement with specific transport mechanisms previously demonstrated for other neurotropic viruses. Cerebrospinal fluid (CSF) represents yet another way in which brain anatomy may be exploited to distribute nanoparticles broadly in the central nervous system. In this study, we assessed the distribution and perivascular transport of nanoparticles of different sizes delivered into the parenchyma of rodents and CSF in non-human primates.