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.

MR-guided delivery of AAV2-BDNF into the entorhinal cortex of non-human primates.

Brain-derived neurotrophic factor (BDNF) gene delivery to the entorhinal cortex is a candidate for treatment of Alzheimer's disease (AD) to reduce neurodegeneration that is associated with memory loss. Accurate targeting of the entorhinal cortex in AD is complex due to the deep and atrophic state of this brain region. Using MRI-guided methods with convection-enhanced delivery, we were able to accurately and consistently target AAV2-BDNF delivery to the entorhinal cortex of non-human primates; 86 ± 3% of transduced cells in the targeted regions co-localized with the neuronal marker NeuN. The volume of AAV2-BDNF (3 × 108 vg/µl) infusion linearly correlated with the number of BDNF labeled cells and the volume (mm3) of BDNF immunoreactivity in the entorhinal cortex. BDNF is normally trafficked to the hippocampus from the entorhinal cortex; in these experiments, we also found that BDNF immunoreactivity was elevated in the hippocampus following therapeutic BDNF vector delivery to the entorhinal cortex, achieving growth factor distribution through key memory circuits. These findings indicate that MRI-guided infusion of AAV2-BDNF to the entorhinal cortex of the non-human primate results in safe and accurate targeting and distribution of BDNF to both the entorhinal cortex and the hippocampus. These methods are adaptable to human clinical trials.

AAV9-mediated expression of a non-self protein in nonhuman primate central nervous system triggers widespread neuroinflammation driven by antigen-presenting cell transduction.

Many studies have demonstrated that adeno-associated virus serotype 9 (AAV9) transduces astrocytes and neurons when infused into rat or nonhuman primate (NHP) brain. We previously showed in rats that transduction of antigen-presenting cells (APC) by AAV9 encoding a foreign protein triggered a full neurotoxic immune response. Accordingly, we asked whether this phenomenon occurred in NHP. We performed parenchymal or intrathecal infusion of AAV9 encoding green fluorescent protein (GFP), a non-self protein derived from jellyfish, or human aromatic L-amino acid decarboxylase (hAADC), a self-protein, in separate NHP. Animals receiving AAV9-GFP into cisterna magna (CM) became ataxic, indicating cerebellar pathology, whereas AAV9-hAADC animals remained healthy. In transduced regions, AAV9-GFP elicited inflammation associated with early activation of astrocytic and microglial cells, along with upregulation of major histocompatibility complex class II (MHC-II) in glia. In addition, we found Purkinje neurons lacking calbindin after AAV9-GFP but not after AAV9-hAADC delivery. Our results demonstrate that AAV9-mediated expression of a foreign-protein, but not self-recognized protein, triggers complete immune responses in NHP regardless of the route of administration. Our results warrant caution when contemplating use of serotypes that can transduce APC if the transgene is not syngeneic with the host. This finding has the potential to complicate preclinical toxicology studies in which such vectors encoding human cDNA's are tested in animals.

Novel platform for MRI-guided convection-enhanced delivery of therapeutics: preclinical validation in nonhuman primate brain.

BACKGROUND/AIMS: A skull-mounted aiming device and integrated software platform has been developed for MRI-guided neurological interventions. In anticipation of upcoming gene therapy clinical trials, we adapted this device for real-time convection-enhanced delivery of therapeutics via a custom-designed infusion cannula. The targeting accuracy of this delivery system and the performance of the infusion cannula were validated in nonhuman primates. METHODS: Infusions of gadoteridol were delivered to multiple brain targets and the targeting error was determined for each cannula placement. Cannula performance was assessed by analyzing gadoteridol distributions and by histological analysis of tissue damage. RESULTS: The average targeting error for all targets (n = 11) was 0.8 mm (95% CI = 0.14). For clinically relevant volumes, the distribution volume of gadoteridol increased as a linear function (R(2) = 0.97) of the infusion volume (average slope = 3.30, 95% CI = 0.2). No infusions in any target produced occlusion, cannula reflux or leakage from adjacent tracts, and no signs of unexpected tissue damage were observed. CONCLUSIONS: This integrated delivery platform allows real-time convection-enhanced delivery to be performed with a high level of precision, predictability and safety. This approach may improve the success rate for clinical trials involving intracerebral drug delivery by direct infusion.

Ultrasound-enhanced drug transport and distribution in the brain.

Drug delivery in the brain is limited by slow drug diffusion in the brain tissue. This study tested the hypothesis that ultrasound can safely enhance the permeation of drugs in the brain. In vitro exposure to ultrasound at various frequencies (85 kHz, 174 kHz, and 1 MHz) enhanced the permeation of tritium-labeled molecules with molecular weight up to 70 kDa across porcine brain tissue. A maximum enhancement of 24-fold was observed at 85 kHz and 1,200 J/cm(2). In vivo exposure to 1-MHz ultrasound further demonstrated the ability of ultrasound to facilitate molecule distribution in the brain of a non-human primate. Finally, ultrasound under conditions similar to those used in vivo was shown to cause no damage to plasmid DNA, siRNA, adeno-associated virus, and fetal rat cortical neurons over a range of conditions. Altogether, these studies demonstrate that ultrasound can increase drug permeation in the brain in vitro and in vivo under conditions that did not cause detectable damage.

Non-PEGylated liposomes for convection-enhanced delivery of topotecan and gadodiamide in malignant glioma: initial experience.

Convection-enhanced delivery (CED) of highly stable PEGylated liposomes encapsulating chemotherapeutic drugs has previously been effective against malignant glioma xenografts. We have developed a novel, convectable non-PEGylated liposomal formulation that can be used to encapsulate both the topoisomerase I inhibitor topotecan (topoCED) and paramagnetic gadodiamide (gadoCED), providing an ideal basis for real-time monitoring of drug distribution. Tissue retention of topoCED following single CED administration was significantly improved relative to free topotecan. At a dose of 10 microg (0.5 mg/ml), topoCED had a half-life in brain of approximately 1 day and increased the area under the concentration-time curve (AUC) by 28-fold over free topotecan (153.8 vs. 5.5 microg day/g). The combination of topoCED and gadoCED was found to co-convect well in both naïve rat brain and malignant glioma xenografts (correlation coefficients 0.97-0.99). In a U87MG cell assay, the 50% inhibitory concentration (IC(50)) of topoCED was approximately 0.8 microM at 48 and 72 h; its concentration-time curves were similar to free topotecan and unaffected by gadoCED. In a U87MG intracranial rat xenograft model, a two-dose CED regimen of topoCED co-infused with gadoCED greatly increased median overall survival at dose levels of 0.5 mg/ml (29.5 days) and 1.0 mg/ml (33.0 days) vs. control (20.0 days; P < 0.0001 for both comparisons). TopoCED at higher concentrations (1.6 mg/ml) co-infused with gadoCED showed no evidence of histopathological changes attributable to either agent. The positive results of tissue pharmacokinetics, co-convection, cytotoxicity, efficacy, and lack of toxicity of topoCED in a clinically meaningful dose range, combined with an ideal matched-liposome paramagnetic agent, gadoCED, implicates further clinical applications of this therapy in the treatment of malignant glioma.

Development of a novel frameless skull-mounted ball-joint guide array for use in image-guided neurosurgery.

OBJECTIVE: Successful convection-enhanced delivery of therapeutic agents to subcortical brain structures requires accurate cannula placement. Stereotactic guiding devices have been developed to accurately target brain nuclei. However, technologies remain limited by a lack of MRI compatibility, or by devices' size, making them suboptimal for direct gene delivery to brain parenchyma. The goal of this study was to validate the accuracy of a novel frameless skull-mounted ball-joint guide array (BJGA) in targeting the nonhuman primate (NHP) brain. METHODS: Fifteen MRI-guided cannula insertions were performed on 9 NHPs, each targeting the putamen. Optimal trajectories were planned on a standard MRI console using 3D multiplanar baseline images. After cannula insertion, the intended trajectory was compared to the final trajectory to assess deviation (euclidean error) of the cannula tip. RESULTS: The average cannula tip deviation was 1.18 ± 0.60 mm (mean ± SD) as measured by 2 independent reviewers. Topological analysis showed a superior, posterior, and rightward directional bias, and the intra- and interclass correlation coefficients were > 0.85, indicating valid and reliable intra- and interobserver evaluation. CONCLUSIONS: The data demonstrate that the BJGA can be used to reliably target subcortical brain structures by using MRI guidance, with accuracy comparable to current frameless stereotactic systems. The size and versatility of the BJGA, combined with a streamlined workflow, allows for its potential applicability to a variety of intracranial neurosurgical procedures, and for greater flexibility in executing MRI-guided experiments within the NHP brain.

Infuse-as-you-go convective delivery to enhance coverage of elongated brain targets: technical note.

OBJECTIVE: To develop and assess a convective delivery technique that enhances the effectiveness of drug delivery to nonspherical brain nuclei, the authors developed an occipital "infuse-as-you-go" approach to the putamen and compared it to the currently used transfrontal approach. METHODS: Eleven nonhuman primates received a bilateral putamen injection of adeno-associated virus with 2 mM gadolinium-DTPA by real-time MR-guided convective perfusion via either a transfrontal (n = 5) or occipital infuse-as-you-go (n = 6) approach. RESULTS: MRI provided contemporaneous assessment and monitoring of putaminal infusions for transfrontal (2 to 3 infusion deposits) and occipital infuse-as-you-go (stepwise infusions) putaminal approaches. The infuse-as-you-go technique was more efficient than the transfrontal approach (mean 35 ± 1.1 vs 88 ± 8.3 minutes [SEM; p < 0.001]). More effective perfusion of the postcommissural and total putamen was achieved with the infuse-as-you-go versus transfronatal approaches (100-µl infusion volumes; mean posterior commissural coverage 76.2% ± 5.0% vs 32.8% ± 2.9% [p < 0.001]; and mean total coverage 53.5% ± 3.0% vs 38.9% ± 2.3% [p < 0.01]). CONCLUSIONS: The infuse-as-you-go approach, paralleling the longitudinal axis of the target structure, provides a more effective and efficient method for convective infusate coverage of elongated, irregularly shaped subcortical brain nuclei.

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.

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.

Canine model of convection-enhanced delivery of liposomes containing CPT-11 monitored with real-time magnetic resonance imaging: laboratory investigation.

OBJECT: Many factors relating to the safety and efficacy of convection-enhanced delivery (CED) into intracranial tumors are poorly understood. To investigate these factors further and establish a more clinically relevant large animal model, with the potential to investigate CED in large, spontaneous tumors, the authors developed a magnetic resonance (MR) imaging-compatible system for CED of liposomal nanoparticles into the canine brain, incorporating real-time MR imaging. Additionally any possible toxicity of liposomes containing Gd and the chemotherapeutic agent irinotecan (CPT-11) was assessed following direct intraparenchymal delivery. METHODS: Four healthy laboratory dogs were infused with liposomes containing Gd, rhodamine, or CPT-11. Convection-enhanced delivery was monitored in real time by sequential MR imaging, and the volumes of distribution were calculated from MR images and histological sections. Assessment of any toxicity was based on clinical and histopathological evaluation. Convection-enhanced delivery resulted in robust volumes of distribution in both gray and white matter, and real-time MR imaging allowed accurate calculation of volumes and pathways of distribution. RESULTS: Infusion variability was greatest in the gray matter, and was associated with leakage into ventricular or subarachnoid spaces. Complications were minimal and included mild transient proprioceptive deficits, focal hemorrhage in 1 dog, and focal, mild perivascular, nonsuppurative encephalitis in 1 dog. CONCLUSIONS: Convection-enhanced delivery of liposomal Gd/CPT-11 is associated with minimal adverse effects in a large animal model, and further assessment for use in clinical patients is warranted. Future studies investigating real-time monitored CED in spontaneous gliomas in canines are feasible and will provide a unique, clinically relevant large animal translational model for testing this and other therapeutic strategies.

Convection-enhanced delivery of tumor necrosis factor-related apoptosis-inducing ligand with systemic administration of temozolomide prolongs survival in an intracranial glioblastoma xenograft model.

Although tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) is a potent activator of cell death, preferentially killing neoplastic cells over normal cells, the efficacy of TRAIL for the treatment of glioma might be limited due to cellular resistance and, importantly, poor distribution after systemic administration. TRAIL and temozolomide (TMZ) were recently shown to have a synergistic antitumor effect against U87MG glioma cells in vitro. Convection-enhanced delivery (CED) can effectively distribute TRAIL protein throughout a brain tumor mass. In this study, we evaluated CED of TRAIL, alone and in conjunction with systemic TMZ administration, for antitumor efficacy. CED of TRAIL demonstrated safe and effective distribution in both normal brain and a U87MG intracranial xenograft model. Individually, both CED of TRAIL and systemic TMZ administration prolonged survival in tumor-bearing rats. However, the combination of these two treatments was significantly more effective than either treatment alone. CED of TRAIL in conjunction with systemic TMZ treatment is a promising strategy for the treatment of malignant gliomas.

Distribution of liposomes into brain and rat brain tumor models by convection-enhanced delivery monitored with magnetic resonance imaging.

Although liposomes have been used as a vehicle for delivery of therapeutic agents in oncology, their efficacy in targeting brain tumors has been limited due to poor penetration through the blood-brain barrier. Because convection-enhanced delivery (CED) of liposomes may improve the therapeutic index for targeting brain tumors, we conducted a three-stage study: stage 1 established the feasibility of using in vivo magnetic resonance imaging (MRI) to confirm adequate liposomal distribution within targeted regions in normal rat brain. Liposomes colabeled with gadolinium (Gd) and a fluorescent indicator, 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine-5,5'-disulfonic acid [DiI-DS; formally DiIC(18)(3)-DS], were administered by CED into striatal regions. The minimum concentration of Gd needed for monitoring, correlation of infused volume with distribution volume, clearance of infused liposome containing Gd and DiI-DS (Lip/Gd/DiI-DS), and potential local toxicity were evaluated. After determination of adequate conditions for MRI detection in normal brain, stage 2 evaluated the feasibility of in vivo MRI monitoring of liposomal distribution in C6 and 9L-2 rat glioma models. In both models, the distribution of Lip/Gd/DiI-DS covering the tumor mass was well defined and monitored with MRI. Stage 3 was designed to develop a clinically relevant treatment strategy in the 9L-2 model by infusing liposome containing Gd (Lip/Gd), prepared in the same size as Lip/Gd/DiI-DS, with Doxil, a liposomal drug of similar size used to treat several cancers. MRI detection of Lip/Gd coadministered with Doxil provided optimum CED parameters for complete coverage of 9L-2 tumors. By permitting in vivo monitoring of therapeutic distribution in brain tumors, this technique optimizes local drug delivery and may provide a basis for clinical applications in the treatment of malignant glioma.

Strong cortical and spinal cord transduction after AAV7 and AAV9 delivery into the cerebrospinal fluid of nonhuman primates.

The present study builds on previous work showing that infusion of adeno-associated virus type 9 (AAV9) into the cisterna magna (CM) of nonhuman primates resulted in widespread transduction throughout cortex and spinal cord. Transduction efficiency was severely limited, however, by the presence of circulating anti-AAV antibodies. Accordingly, we compared AAV9 to a related serotype, AAV7, which has a high capsid homology. CM infusion of either AAV7 or AAV9 directed high level of cell transduction with similar patterns of distribution throughout brain cortex and along the spinal cord. Dorsal root ganglia and corticospinal tracts were also transduced. Both astrocytes and neurons were transduced. Interestingly, little transduction was observed in peripheral organs. Our results indicate that intrathecal delivery of either AAV7 or AAV9 directs a robust and widespread cellular transduction in the central nervous system and other peripheral neural structures.

Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates.

Widespread distribution of gene products at clinically relevant levels throughout the CNS has been challenging. Adeno-associated virus type 9 (AAV9) vector has been reported as a good candidate for intravascular gene delivery, but low levels of preexisting antibody titers against AAV in the blood abrogate cellular transduction within the CNS. In the present study we compared the effectiveness of vascular delivery and cerebrospinal fluid (CSF) delivery of AAV9 in transducing CNS tissue in nonhuman primates. Both delivery routes generated similar distribution patterns, although we observed a more robust level of transduction after CSF delivery. Consistent with previous reports administering AAV9, we found greater astrocytic than neuronal tropism via both routes, although we did find a greater magnitude of CNS transduction after CSF delivery compared with intravascular delivery. Last, we have demonstrated that delivery of AAV9 into the CSF does not shield against AAV antibodies. This has obvious implications when developing and/or implementing any clinical trial studies.

Canine spontaneous glioma: a translational model system for convection-enhanced delivery.

Canine spontaneous intracranial tumors bear striking similarities to their human tumor counterparts and have the potential to provide a large animal model system for more realistic validation of novel therapies typically developed in small rodent models. We used spontaneously occurring canine gliomas to investigate the use of convection-enhanced delivery (CED) of liposomal nanoparticles, containing topoisomerase inhibitor CPT-11. To facilitate visualization of intratumoral infusions by real-time magnetic resonance imaging (MRI), we included identically formulated liposomes loaded with Gadoteridol. Real-time MRI defined distribution of infusate within both tumor and normal brain tissues. The most important limiting factor for volume of distribution within tumor tissue was the leakage of infusate into ventricular or subarachnoid spaces. Decreased tumor volume, tumor necrosis, and modulation of tumor phenotype correlated with volume of distribution of infusate (Vd), infusion location, and leakage as determined by real-time MRI and histopathology. This study demonstrates the potential for canine spontaneous gliomas as a model system for the validation and development of novel therapeutic strategies for human brain tumors. Data obtained from infusions monitored in real time in a large, spontaneous tumor may provide information, allowing more accurate prediction and optimization of infusion parameters. Variability in Vd between tumors strongly suggests that real-time imaging should be an essential component of CED therapeutic trials to allow minimization of inappropriate infusions and accurate assessment of clinical outcomes.

Gadolinium-loaded liposomes allow for real-time magnetic resonance imaging of convection-enhanced delivery in the primate brain.

Drug delivery to brain tumors has long posed a major challenge. Convection-enhanced delivery (CED) has been developed as a drug delivery strategy to overcome this difficulty. Ideally, direct visualization of the tissue distribution of drugs infused by CED would assure successful delivery of therapeutic agents to the brain tumor while minimizing exposure of the normal brain. We previously developed a magnetic resonance imaging (MRI)-based method to visualize the distribution of liposomal agents after CED in rodent brains. In the present study, CED of liposomes was further examined in the non-human primate brain (n = 6). Liposomes containing Gadoteridol, DiI-DS, and rhodamine were infused in corona radiata, putamen nucleus, and brain stem. Volume of distribution was analyzed for all delivery locations by histology and MR imaging. Real-time MRI monitoring of liposomes containing gadolinium allowed direct visualization of a robust distribution. MRI of liposomal gadolinium was highly accurate at determining tissue distribution, as confirmed by comparison with histological results from concomitant administration of fluorescent liposomes. Linear correlation for liposomal infusions between infusion volume and distribution volume was established in all targeted locations. We conclude that an integrated strategy combining liposome/nanoparticle technology, CED, and MRI may provide new opportunities for the treatment of brain tumors. Our ability to directly monitor and to control local delivery of liposomal drugs will most likely result in greater clinical efficacy when using CED in management of patients.

Extensive distribution of liposomes in rodent brains and brain tumors following convection-enhanced delivery.

Liposomes labeled with various markers were subjected to local-regional administration with either direct injection or convection-enhanced delivery (CED) into rodent brains and brain tumor models. Direct injection of liposomes containing attached or encapsulated fluorochromes and/or encapsulated gold particles indicated that tissue localization of liposomes could be sensitively and specifically detected in the central nervous system (CNS). When CED was applied, liposomes achieved extensive and efficient distribution within normal mouse brains. Co-infusion of mannitol further increased tissue penetration of liposomes. Liposomes were also loaded with gadodiamide to monitor their CNS distribution in rats by magnetic resonance imaging (MRI). CED-infused liposomes were readily seen on MRI scans as large regions of intense signal at 2 h, and more diffuse regions at 24 h. Finally, labeled liposomes were infused via CED into tumor tissue in glioma xenograft models in rodent hosts. In intracranial U-87 glioma xenografts, CED-infused liposomes had distributed throughout tumor tissue, including extension into surrounding normal tissue. Greater penetration was observed using 40 versus 90 nm liposomes, as well as with mannitol co-infusion. To our knowledge, this is the first report of CED infusion of liposomes into the CNS. We conclude that CED of liposomes in the CNS is a feasible approach, and offers a promising strategy for targeting therapeutic agents to brain tumors.