Safety and efficacy of cerliponase alfa in children with neuronal ceroid lipofuscinosis type 2 (CLN2 disease): an open-label extension study.

BACKGROUND: Cerliponase alfa is a recombinant human tripeptidyl peptidase 1 (TPP1) enzyme replacement therapy for the treatment of neuronal ceroid lipofuscinosis type 2 (CLN2 disease), which is caused by mutations in the TPP1 gene. We aimed to determine the long-term safety and efficacy of intracerebroventricular cerliponase alfa in children with CLN2 disease. METHODS: This analysis includes cumulative data from a primary 48-week, single-arm, open-label, multicentre, dose-escalation study (NCT01907087) and the 240-week open-label extension with 6-month safety follow-up, conducted at five hospitals in Germany, Italy, the UK, and the USA. Children aged 3-16 years with CLN2 disease confirmed by genetic analysis and enzyme testing were eligible for inclusion. Treatment was intracerebroventricular infusion of 300 mg cerliponase alfa every 2 weeks. Historical controls with untreated CLN2 disease in the DEM-CHILD database were used as a comparator group. The primary efficacy outcome was time to an unreversed 2-point decline or score of 0 in the combined motor and language domains of the CLN2 Clinical Rating Scale. This extension study is registered with ClinicalTrials.gov, NCT02485899, and is complete. FINDINGS: Between Sept 13, 2013, and Dec 22, 2014, 24 participants were enrolled in the primary study (15 female and 9 male). Of those, 23 participants were enrolled in the extension study, conducted between Feb 2, 2015, and Dec 10, 2020, and received 300 mg cerliponase alfa for a mean of 272·1 (range 162·1-300·1) weeks. 17 participants completed the extension and six discontinued prematurely. Treated patients were significantly less likely than historical untreated controls to have an unreversed 2-point decline or score of 0 in the combined motor and language domains (hazard ratio 0·14, 95% CI 0·06 to 0·33; p<0·0001). All participants experienced at least one adverse event and 21 (88%) experienced a serious adverse event; nine participants experienced intracerebroventricular device-related infections, with nine events in six participants resulting in device replacement. There were no study discontinuations because of an adverse event and no deaths. INTERPRETATION: Cerliponase alfa over a mean treatment period of more than 5 years was seen to confer a clinically meaningful slowing of decline of motor and language function in children with CLN2 disease. Although our study does not have a contemporaneous control group, the results provide crucial insights into the effects of long-term treatment. FUNDING: BioMarin Pharmaceutical.

Positron Emission Tomography Quantitative Assessment of Off-Target Whole-Body Biodistribution of I-124-Labeled Adeno-Associated Virus Capsids Administered to Cerebral Spinal Fluid.

Based on studies in experimental animals demonstrating that administration of adeno-associated virus (AAV) vectors to the cerebrospinal fluid (CSF) is an effective route to transfer genes to the nervous system, there are increasing number of clinical trials using the CSF route to treat nervous system disorders. With the knowledge that the CSF turns over four to five times daily, and evidence in experimental animals that at least some of CSF administered AAV vectors are distributed to systemic organs, we asked: with AAV administration to the CSF, what fraction of the total dose remains in the nervous system and what fraction goes off target and is delivered systemically? To quantify the biodistribution of AAV capsids immediately after administration, we covalently labeled AAV capsids with iodine 124 (I-124), a cyclotron generated positron emitter, enabling quantitative positron emission tomography scanning of capsid distribution for up to 96 h after AAV vector administration. We assessed the biodistribution to nonhuman primates of I-124-labeled capsids from different AAV clades, including 9 (clade F), rh.10 (E), PHP.eB (F), hu68 (F), and rh91(A). The analysis demonstrated that 60-90% of AAV vectors administered to the CSF through either the intracisternal or intrathecal (lumbar) routes distributed systemically to major organs. These observations have potentially significant clinical implications regarding accuracy of AAV vector dosing to the nervous system, evoking systemic immunity at levels similar to that with systemic administration, and potential toxicity of genes designed to treat nervous system disorders being expressed in non-nervous system organs. Based on these data, individuals in clinical trials using AAV vectors administered to the CSF should be monitored for systemic as well as nervous system adverse events and CNS dosing considerations should account for a significant AAV systemic distribution.

A pathway towards a two-dimensional, bore-mounted, volume body coil concept for ultra high-field magnetic resonance imaging.

Lack of a body-sized, bore-mounted, radiofrequency (RF) body coil for ultrahigh field (UHF) magnetic resonance imaging (MRI) is one of the major drawbacks of UHF, hampering the clinical potential of the technology. Transmit field (B1 ) nonuniformity and low specific absorption rate (SAR) efficiencies in UHF MRI are two challenges to be overcome. To address these problems, and ultimately provide a pathway for the full clinical potential of the modality, we have designed and simulated two-dimensional cylindrical high-pass ladder (2D c-HPL) architectures for clinical bore-size dimensions, and demonstrated a simplified proof of concept with a head-sized prototype at 7 T. A new dispersion relation has been derived and electromagnetic simulations were used to verify coil modes. The coefficient of variation (CV) for brain, cerebellum, heart, and prostate tissues after B1 + shimming in silico is reported and compared with previous works. Three prototypes were designed in simulation: a head-sized, body-sized, and long body-sized coil. The head-sized coil showed a CV of 12.3%, a B1 + efficiency of 1.33 µT/√W, and a SAR efficiency of 2.14 µT/√(W/kg) for brain simulations. The body-sized 2D c-HPL coil was compared with same-sized transverse electromagnetic (TEM) and birdcage coils in silico with a four-port circularly polarized mode excitation. Improved B1 + uniformity (26.9%) and SAR efficiency (16% and 50% better than birdcage and TEM coils, respectively) in spherical phantoms was observed. We achieved a CV of 12.3%, 4.9%, 16.7%, and 2.8% for the brain, cerebellum, heart, and prostate, respectively. Preliminary imaging results for the head-sized coil show good agreement between simulation and experiment. Extending the 1D birdcage coil concept to 2D c-HPLs provides improved B1 + uniformity and SAR efficiency.

Use of small animal PET-CT imaging for in vivo assessment of tendon-to-bone healing: A pilot study.

BACKGROUND: The availability of non-invasive means to evaluate and monitor tendon-bone healing processes in-vivo is limited. Micro Positron-Emission-Tomography (µPET) using 18F-Fluoride is a minimally invasive imaging modality, with which osteoblast activity and bone turnover can be assessed. The aim of this study was to investigate the use of serial in-vivo µPET/CT scans to evaluate bone turnover along the graft-tunnel interface in a rat ACL (anterior cruciate ligament) reconstruction model. METHODS: Unilateral autograft ACL reconstruction was performed in six rats. µPET/CT-scans using 18F-Fluoride were performed 7, 14, 21, and 28 days postoperatively. Standard uptake values (SUV) were calculated for three tunnel regions (intraarticular aperture (IAA), mid-tunnel, and extraarticular aperture (EAA)) of the proximal tibia. Animals were sacrificed at 28 days and evaluated with µCT and histological analysis. RESULTS: SUVs in both bone tunnels showed an increased 18F-Fluoride uptake at 7 days when compared to 14, 21, and 28 days. SUVs showed a gradient on the tibial side, with most bone turnover in the IAA and least in the EAA. At 7, 14, 21, and 28 days, there were significantly higher SUV values in the IAA compared to the EAA (p = .01, < .01, < .01, < .01). SUVs positively correlated with new bone volumetric density obtained with µCT (r = 0.449, p = .013). Volumetric density of newly formed bone detected on µCT correlated with osteoblast numbers observed along the tunnels in histological sections (r = 0.452, p < .016). CONCLUSIONS: Serial in-vivo µPET/CT-scanning has the potential to provide insight into bone turnover and therefore osteoblastic activity during the healing process. As a result, it allows us to directly measure the effect of interventional strategies in tendon-bone healing.

Safety of Direct Intraparenchymal AAVrh.10-Mediated Central Nervous System Gene Therapy for Metachromatic Leukodystrophy.

Metachromatic leukodystrophy, a fatal pediatric neurodegenerative lysosomal storage disease caused by mutations in the arylsulfatase A (ARSA) gene, is characterized by intracellular accumulation of sulfatides in the lysosomes of cells of the central nervous system (CNS). In previous studies, we have demonstrated efficacy of AAVrh.10hARSA, an adeno-associated virus (AAV) serotype rh.10 vector coding for the human ARSA gene to the CNS of a mouse model of the disease, and that catheter-based intraparenchymal administration of AAVrh.10hARSA to the CNS of nonhuman primates (NHPs) white matter results in widespread expression of ARSA. As a formal dose-escalating safety/toxicology study, we assessed the safety of intraparenchymal delivery of AAVrh.10hARSA vector to 12 sites in the white matter of the CNS of NHPs at 2.85 × 1010 (total low dose, 2.4 × 109 genome copies [gc]/site) and 1.5 × 1012 (total high dose, 1.3 × 1011 gc/site) gc, compared to AAVrh.10Null (1.5 × 1012 gc total, 1.3 × 1011 gc/site) as a vector control, and phosphate buffered saline for a sham surgical control. No significant adverse effects were observed in animals treated with low dose AAVrh.10hARSA. However, animals treated with the high dose AAVrh.10ARSA and the high dose Null vector had highly localized CNS abnormalities on magnetic resonance imaging scans at the sites of catheter infusions, and histopathology demonstrated that these sites were associated with infiltrates of T cells, B cells, microglial cells, and/or macrophages. Although these findings had no clinical consequences, these safety data contribute to understanding the dose limits for CNS white matter direct intraparenchymal administration of AAVrh.10 vectors for treatment of CNS disorders.

Slowing late infantile Batten disease by direct brain parenchymal administration of a rh.10 adeno-associated virus expressing CLN2.

Late infantile Batten disease (CLN2 disease) is an autosomal recessive, neurodegenerative lysosomal storage disease caused by mutations in the CLN2 gene encoding tripeptidyl peptidase 1 (TPP1). We tested intraparenchymal delivery of AAVrh.10hCLN2, a nonhuman serotype rh.10 adeno-associated virus vector encoding human CLN2, in a nonrandomized trial consisting of two arms assessed over 18 months: AAVrh.10hCLN2-treated cohort of 8 children with mild to moderate disease and an untreated, Weill Cornell natural history cohort consisting of 12 children. The treated cohort was also compared to an untreated European natural history cohort of CLN2 disease. The vector was administered through six burr holes directly to 12 sites in the brain without immunosuppression. In an additional safety assessment under a separate protocol, five children with severe CLN2 disease were treated with AAVrh.10hCLN2. The therapy was associated with a variety of expected adverse events, none causing long-term disability. Induction of systemic anti-AAVrh.10 immunity was mild. After therapy, the treated cohort had a 1.3- to 2.6-fold increase in cerebral spinal fluid TPP1. There was a slower loss of gray matter volume in four of seven children by MRI and a 42.4 and 47.5% reduction in the rate of decline of motor and language function, compared to Weill Cornell natural history cohort (P < 0.04) and European natural history cohort (P < 0.0001), respectively. Intraparenchymal brain administration of AAVrh.10hCLN2 slowed the progression of disease in children with CLN2 disease. However, improvements in vector design and delivery strategies will be necessary to halt disease progression using gene therapy.

Quantitative Whole-Body Imaging of I-124-Labeled Adeno-Associated Viral Vector Biodistribution in Nonhuman Primates.

A method is presented for quantitative analysis of the biodistribution of adeno-associated virus (AAV) gene transfer vectors following in vivo administration. We used iodine-124 (I-124) radiolabeling of the AAV capsid and positron emission tomography combined with compartmental modeling to quantify whole-body and organ-specific biodistribution of AAV capsids from 1 to 72 h following administration. Using intravenous (IV) and intracisternal (IC) routes of administration of AAVrh.10 and AAV9 vectors to nonhuman primates in the absence or presence of anticapsid immunity, we have identified novel insights into initial capsid biodistribution and organ-specific capsid half-life. Neither I-124-labeled AAVrh.10 nor AAV9 administered intravenously was detected at significant levels in the brain relative to the administered vector dose. Approximately 50% of the intravenously administered labeled capsids were dispersed throughout the body, independent of the liver, heart, and spleen. When administered by the IC route, the labeled capsid had a half-life of ∼10 h in the cerebral spinal fluid (CSF), suggesting that by this route, the CSF serves as a source with slow diffusion into the brain. For both IV and IC administration, there was significant influence of pre-existing anticapsid immunity on I-124-capsid biodistribution. The methodology facilitates quantitative in vivo viral vector dosimetry, which can serve as a technique for evaluation of both on- and off-target organ biodistribution, and potentially accelerate gene therapy development through rapid prototyping of novel vector designs.

Untargeted Metabolite Profiling of Cerebrospinal Fluid Uncovers Biomarkers for Severity of Late Infantile Neuronal Ceroid Lipofuscinosis (CLN2, Batten Disease).

Late infantile neuronal ceroid lipofuscinosis (CLN2 disease) is a rare lysosomal storage disorder caused by a monogenetic deficiency of tripeptidyl peptidase-1 (TPP1). Despite knowledge that lipofuscin is the hallmark disease product, the relevant TPP1 substrate and its role in neuronal physiology/pathology is unknown. We hypothesized that untargeted metabolite profiling of cerebrospinal fluid (CSF) could be used as an effective tool to identify disease-associated metabolic disruptions in CLN2 disease, offering the potential to identify biomarkers that inform on disease severity and progression. Accordingly, a mass spectrometry-based untargeted metabolite profiling approach was employed to differentiate CSF from normal vs. CLN2 deficient individuals. Of 1,433 metabolite features surveyed, 29 linearly correlated with currently employed disease severity scores. With tandem mass spectrometry 8 distinct metabolite identities were structurally confirmed based on retention time and fragmentation pattern matches, vs. standards. These putative CLN2 biomarkers include 7 acetylated species - all attenuated in CLN2 compared to controls. Because acetate is the major bioenergetic fuel for support of mitochondrial respiration, deficient acetylated species in CSF suggests a brain energy defect that may drive neurodegeneration. Targeted analysis of these metabolites in CSF of CLN2 patients offers a powerful new approach for monitoring CLN2 disease progression and response to therapy.

Safe and stable noninvasive focal gene delivery to the mammalian brain following focused ultrasound.

OBJECTIVE: Surgical infusion of gene therapy vectors has provided opportunities for biological manipulation of specific brain circuits in both animal models and human patients. Transient focal opening of the blood-brain barrier (BBB) by MR-guided focused ultrasound (MRgFUS) raises the possibility of noninvasive CNS gene therapy to target precise brain regions. However, variable efficiency and short follow-up of studies to date, along with recent suggestions of the potential for immune reactions following MRgFUS BBB disruption, all raise questions regarding the viability of this approach for clinical translation. The objective of the current study was to evaluate the efficiency, safety, and long-term stability of MRgFUS-mediated noninvasive gene therapy in the mammalian brain. METHODS: Focused ultrasound under the control of MRI, in combination with microbubbles consisting of albumin-coated gas microspheres, was applied to rat striatum, followed by intravenous infusion of an adeno-associated virus serotype 1/2 (AAV1/2) vector expressing green fluorescent protein (GFP) as a marker. Following recovery, animals were followed from several hours up to 15 months. Immunostaining for GFP quantified transduction efficiency and stability of expression. Quantification of neuronal markers was used to determine histological safety over time, while inflammatory markers were examined for evidence of immune responses. RESULTS: Transitory disruption of the BBB by MRgFUS resulted in efficient delivery of the AAV1/2 vector to the targeted rodent striatum, with 50%-75% of striatal neurons transduced on average. GFP transgene expression appeared to be stable over extended periods of time, from 2 weeks to 6 months, with evidence of ongoing stable expression as long as 16 months in a smaller cohort of animals. No evidence of substantial toxicity, tissue injury, or neuronal loss was observed. While transient inflammation from BBB disruption alone was noted for the first few days, consistent with prior observations, no evidence of brain inflammation was observed from 2 weeks to 6 months following MRgFUS BBB opening, despite delivery of a virus and expression of a foreign protein in target neurons. CONCLUSIONS: This study demonstrates that transitory BBB disruption using MRgFUS can be a safe and efficient method for site-specific delivery of viral vectors to the brain, raising the potential for noninvasive focal human gene therapy for neurological disorders.

AAVrh.10-Mediated APOE2 Central Nervous System Gene Therapy for APOE4-Associated Alzheimer's Disease.

Alzheimer's disease (AD) is a progressive degenerative neurological disorder affecting nearly one in nine elderly people in the United States. Population studies have shown that an inheritance of the apolipoprotein E (APOE) variant APOE4 allele increases the risk of developing AD, whereas APOE2 homozygotes are protected from late-onset AD. It was hypothesized that expression of the "protective" APOE2 variant by genetic modification of the central nervous system (CNS) of APOE4 homozygotes could reverse or prevent progressive neurologic damage. To assess the CNS distribution and safety of APOE2 gene therapy for AD in a large-animal model, intraparenchymal, intracisternal, and intraventricular routes of delivery to the CNS of nonhuman primates of AAVrh.10hAPOE2-HA, an AAVrh.10 serotype coding for an HA-tagged human APOE2 cDNA sequence, were evaluated. To evaluate the route of delivery that achieves the widest extent of APOE2 expression in the CNS, the expression of APOE2 in the CNS was evaluated 2 months following vector administration for APOE2 DNA, mRNA, and protein. Finally, using conventional toxicology assays, the safety of the best route of delivery was assessed. The data demonstrated that while all three routes are capable of mediating ApoE2 expression in AD relevant regions, intracisternal delivery of AAVrh.10hAPOE2-HA safely mediated wide distribution of ApoE2 with the least invasive surgical intervention, thus providing the optimal strategy to deliver vector-mediated human APOE2 to the CNS.

Clinical improvement associated with targeted interruption of the cerebellothalamic tract following MR-guided focused ultrasound for essential tremor.

OBJECTIVE The objective of this study was to evaluate the utility of diffusion tensor imaging (DTI) tractography-based targeting of the dentatorubrothalamic tract (DRT) for magnetic resonance-guided focused ultrasound (MRgFUS) thalamotomy in patients with essential tremor (ET) and correlate postprocedural tract disruption with clinical outcomes. METHODS Four patients received preprocedural and immediate postprocedural DTI in addition to traditional anatomical MRI sequences for MRgFUS thalamotomy. Optimal ablation sites were selected based on the patient-specific location of the DRT as demonstrated by DTI (direct targeting) and correlated with traditional atlas-based measurements for thalamic ventral intermediate nucleus (Vim) lesioning (indirect targeting). Fiber tracts were displayed three-dimensionally during the procedure and used in conjunction with clinical signs of tremor control for fine correction of the ablation site. Immediately following the conclusion of the procedure, the MRgFUS head frame was removed and patients were placed in a 32-channel MRI head coil for follow-up DTI and anatomical MRI sequences. RESULTS All patients had excellent postoperative tremor control and successful pre- and postprocedural DTI fiber tracking of the corticospinal tract, medial lemniscus, and DRT. Immediate postprocedure DTI failed to track the DRT ipsilateral to the lesion site with a preserved contralateral DRT, coincident with substantial resolution of contralateral tremor. CONCLUSIONS DTI can reliably identify the optimal ablation target and demonstrates tract disruption on immediate postprocedural imaging. A clinical improvement of ET was observed immediately following the procedure, correlating with DRT disruption and suggesting that interruption of the DRT is a consequence of clinically successful MRgFUS thalamotomy. These findings may have utility for both MRgFUS procedure planning in surgically naive patients and retreatment of patients who have previously undergone unsuccessful thalamic Vim lesioning.

Magnetic resonance microscopy may enable distinction between normal histomorphological features and prostate cancer in the resected prostate gland.

OBJECTIVES: To determine imaging protocol parameters for characterization of prostate tissue at histological length scales. MATERIAL AND METHODS: Rapid acquisition with relaxation enhancement, spin echo and gradient echo fast low angle shot data were acquired using ex vivo 3-Tesla or 7-Tesla magnetic field strengths from fresh prostatectomy specimens (n = 15) obtained from either organ donor or patients with prostate cancer (PCa). To achieve the closest correspondence between histopathological components and magnetic resonance imaging (MRI) results, in terms of resolution and sectioning planes, multiple high-resolution imaging protocols (ranging from a few minutes to overnight) were tested. Ductograms were generated as part of image post-processing. Specimens were subsequently submitted for histopathological evaluation. RESULTS: A total of seven imaging protocols were tested. Ex vivo 7-Tesla MRI identified normal components of prostate glands, including ducts, blood vessels, concretions and stroma at a spatial resolution of 60 × 60 × 60 µm3 to 107 × 107 × 500 µm3 . Malignant glands and nests of tumour cells identified at 60 × 60 × 90 µm3 were highly similar to low-magnification (×2) histopathology. Ductograms enhanced the differentiation between benign and malignant glands. The results of the present study were encouraging, and further work is warranted with a larger sample size. CONCLUSION: We showed that critical histopathological features of the prostate gland can be identified with high-resolution ex vivo MRI examination and this offers promise that MRI microscopy of PCa will ultimately be possible in vivo.

Magnetic resonance advection imaging of cerebrovascular pulse dynamics.

We analyze the pulsatile signal component of dynamic echo planar imaging data from the brain by modeling the dependence between local temporal and spatial signal variability. The resulting magnetic resonance advection imaging maps depict the location of major arteries. Color direction maps allow for visualization of the direction of blood vessels. The potential significance of magnetic resonance advection imaging maps is demonstrated on a functional magnetic resonance imaging data set of 19 healthy subjects. A comparison with the here introduced pulse coherence maps, in which the echo planar imaging signal is correlated with a cardiac pulse signal, shows that the magnetic resonance advection imaging approach results in a better spatial definition without the need for a pulse reference. In addition, it is shown that magnetic resonance advection imaging velocities can be estimates of pulse wave velocities if certain requirements are met, which are specified. Although for this application magnetic resonance advection imaging velocities are not quantitative estimates of pulse wave velocities, they clearly depict local pulsatile dynamics. Magnetic resonance advection imaging can be applied to existing dynamic echo planar imaging data sets with sufficient spatiotemporal resolution. It is discussed whether magnetic resonance advection imaging might have the potential to evolve into a biomarker for the health of the cerebrovascular system.

The Potential of High Resolution Magnetic Resonance Microscopy in the Pathologic Analysis of Resected Breast and Lymph Tissue.

Pathologic evaluation of breast specimens requires a fixation and staining procedure of at least 12 hours duration, delaying diagnosis and post-operative planning. Here we introduce an MRI technique with a custom-designed radiofrequency resonator for imaging breast and lymph tissue with sufficient spatial resolution and speed to guide pathologic interpretation and offer value in clinical decision making. In this study, we demonstrate the ability to image breast and lymphatic tissue using 7.0 Tesla MRI, achieving a spatial resolution of 59 × 59 × 94 µm(3) with a signal-to-noise ratio of 15-20, in an imaging time of 56 to 70 minutes. These are the first MR images to reveal characteristic pathologic features of both benign and malignant breast and lymph tissue, some of which were discernible by blinded pathologists who had no prior training in high resolution MRI interpretation.

Genetic modification of neurons to express bevacizumab for local anti-angiogenesis treatment of glioblastoma.

The median survival of glioblastoma multiforme (GBM) is approximately 1 year. Following surgical removal, systemic therapies are limited by the blood-brain barrier. To circumvent this, we developed a method to modify neurons with the genetic sequence for therapeutic monoclonal antibodies using adeno-associated virus (AAV) gene transfer vectors, directing persistent, local expression in the tumor milieu. The human U87MG GBM cell line or patient-derived early passage GBM cells were administered to the striatum of NOD/SCID immunodeficient mice. AAVrh.10BevMab, an AAVrh.10-based vector coding for bevacizumab (Avastin), an anti-human vascular endothelial growth factor (VEGF) monoclonal antibody, was delivered to the area of the GBM xenograft. Localized expression of bevacizumab was demonstrated by quantitative PCR, ELISA and western blotting. Immunohistochemistry showed that bevacizumab was expressed in neurons. Concurrent administration of AAVrh.10BevMab with the U87MG tumor reduced tumor blood vessel density and tumor volume, and increased survival. Administration of AAVrh.10BevMab 1 week after U87MG xenograft reduced growth and increased survival. Studies with patient-derived early passage GBM primary cells showed a reduction in primary tumor burden with an increased survival. These data support the strategy of AAV-mediated central nervous system gene therapy to treat GBM, overcoming the blood-brain barrier through local, persistent delivery of an anti-angiogenesis monoclonal antibody.

Intra-arterial delivery of AAV vectors to the mouse brain after mannitol mediated blood brain barrier disruption.

The delivery of therapeutics to neural tissue is greatly hindered by the blood brain barrier (BBB). Direct local delivery via diffusive release from degradable implants or direct intra-cerebral injection can bypass the BBB and obtain high concentrations of the therapeutic in the targeted tissue, however the total volume of tissue that can be treated using these techniques is limited. One treatment modality that can potentially access large volumes of neural tissue in a single treatment is intra-arterial (IA) injection after osmotic blood brain barrier disruption. In this technique, the therapeutic of interest is injected directly into the arteries that feed the target tissue after the blood brain barrier has been disrupted by exposure to a hyperosmolar mannitol solution, permitting the transluminal transport of the therapy. In this work we used contrast enhanced magnetic resonance imaging (MRI) studies of IA injections in mice to establish parameters that allow for extensive and reproducible BBB disruption. We found that the volume but not the flow rate of the mannitol injection has a significant effect on the degree of disruption. To determine whether the degree of disruption that we observed with this method was sufficient for delivery of nanoscale therapeutics, we performed IA injections of an adeno-associated viral vector containing the CLN2 gene (AAVrh.10CLN2), which is mutated in the lysosomal storage disorder Late Infantile Neuronal Ceroid Lipofuscinosis (LINCL). We demonstrated that IA injection of AAVrh.10CLN2 after BBB disruption can achieve widespread transgene production in the mouse brain after a single administration. Further, we showed that there exists a minimum threshold of BBB disruption necessary to permit the AAV.rh10 vector to pass into the brain parenchyma from the vascular system. These results suggest that IA administration may be used to obtain widespread delivery of nanoscale therapeutics throughout the murine brain after a single administration.

Tissue-engineered intervertebral discs: MRI results and histology in the rodent spine.

OBJECT: Tissue-engineered intervertebral discs (TE-IVDs) represent a new experimental approach for the treatment of degenerative disc disease. Compared with mechanical implants, TE-IVDs may better mimic the properties of native discs. The authors conducted a study to evaluate the outcome of TE-IVDs implanted into the rat-tail spine using radiological parameters and histology. METHODS: Tissue-engineered intervertebral discs consist of a distinct nucleus pulposus (NP) and anulus fibrosus (AF) that are engineered in vitro from sheep IVD chondrocytes. In 10 athymic rats a discectomy in the caudal spine was performed. The discs were replaced with TE-IVDs. Animals were kept alive for 8 months and were killed for histological evaluation. At 1, 5, and 8 months, MR images were obtained; T1-weighted sequences were used for disc height measurements, and T2-weighted sequences were used for morphological analysis. Quantitative T2 relaxation time analysis was used to assess the water content and T1ρ-relaxation time to assess the proteoglycan content of TE-IVDs. RESULTS: Disc height of the transplanted segments remained constant between 68% and 74% of healthy discs. Examination of TE-IVDs on MR images revealed morphology similar to that of native discs. T2-relaxation time did not differ between implanted and healthy discs, indicating similar water content of the NP tissue. The size of the NP decreased in TE-IVDs. Proteoglycan content in the NP was lower than it was in control discs. Ossification of the implanted segment was not observed. Histological examination revealed an AF consisting of an organized parallel-aligned fiber structure. The NP matrix appeared amorphous and contained cells that resembled chondrocytes. CONCLUSIONS: The TE-IVDs remained viable over 8 months in vivo and maintained a structure similar to that of native discs. Tissue-engineered intervertebral discs should be explored further as an option for the potential treatment of degenerative disc disease.

Cannulation of the internal carotid artery in mice: a novel technique for intra-arterial delivery of therapeutics.

We have developed a novel minimally invasive technique for the intra-arterial delivery of therapeutics to the mouse brain. CD-1 mice were anesthetized and placed in a lateral decubitus position. A 10mm midline longitudinal incision was made over the thyroid bone. The omohyoid and sternomastoid muscles were retracted to expose the common carotid artery and external carotid artery (ECA). To maximize delivery of administered agents, the superior thyroid artery was ligated or coagulated, and the occipital artery and the pterygopalatine artery (PPA) were temporarily occluded with 6-0 prolene suture. The ECA was carefully dissected and a permanent ligature was placed on its distal segment while a temporary 6-0 prolene ligature was placed on the proximal segment in order to obtain a flow-free segment of vessel. A sterilized 169 µm outer diameter polyimide microcatheter was introduced into the ECA and advanced in retrograde fashion toward the carotid bifurcation. The catheter was then secured and manually rotated so that the microcatheter tip was oriented cephalad in the internal carotid artery (ICA). We were able to achieve reproducible results for selective ipsilateral hemispheric carotid injections of mannitol mediated therapeutics and/or gadolinium-based MRI contrast agent. Survival rates were dependent on the administered agent and ranged from 78 to 90%. This technique allows for reproducible delivery of agents to the ipsilateral cerebral hemisphere by utilizing anterograde catheter placement and temporary ligation of the PPA. This method is cost-effective and associated with a low rate of morbimortality.

Adenovirus capsid-based anti-cocaine vaccine prevents cocaine from binding to the nonhuman primate CNS dopamine transporter.

Cocaine addiction is a major problem for which there is no approved pharmacotherapy. We have developed a vaccine to cocaine (dAd5GNE), based on the cocaine analog GNE linked to the capsid proteins of a serotype 5 adenovirus, designed to evoke anti-cocaine antibodies that sequester cocaine in the blood, preventing access to the CNS. To assess the efficacy of dAd5GNE in a large animal model, positron emission tomography (PET) and the radiotracer [(11)C]PE2I were used to measure cocaine occupancy of the dopamine transporter (DAT) in nonhuman primates. Repeat administration of dAd5GNE induced high anti-cocaine titers. Before vaccination, cocaine displaced PE2I from DAT in the caudate and putamen, resulting in 62±4% cocaine occupancy. In contrast, dAd5GNE-vaccinated animals showed reduced cocaine occupancy such that when anti-cocaine titers were >4 × 10(5), the cocaine occupancy was reduced to levels of <20%, significantly below the 47% threshold required to evoke the subjective 'high' reported in humans.

Orthotopic glioblastoma stem-like cell xenograft model in mice to evaluate intra-arterial delivery of bevacizumab: from bedside to bench.

Bevacizumab (BV), a humanized monocolonal antibody directed against vascular endothelial growth factor (VEGF), is a standard intravenous (IV) treatment for recurrent glioblastoma multiforme (GBM), that has been introduced recently as an intra-arterial (IA) treatment modality in humans. Since preclinical models have not been reported, we sought to develop a tumor stem cell (TSC) xenograft model to investigate IA BV delivery in vivo. Firefly luciferase transduced patient TSC were injected into the cortex of 35 nude mice. Tumor growth was monitored weekly using bioluminescence imaging. Mice were treated with either intraperitoneal (IP) or IA BV, with or without blood-brain barrier disruption (BBBD), or with IP saline injection (controls). Tumor tissue was analyzed using immunohistochemistry and western blot techniques. Tumor formation occurred in 31 of 35 (89%) mice with a significant signal increase over time (p=0.018). Post mortem histology revealed an infiltrative growth of TSC xenografts in a similar pattern compared to the primary human GBM. Tumor tissue analyzed at 24 hours after treatment revealed that IA BV treatment with BBBD led to a significantly higher intratumoral BV concentration compared to IA BV alone, IP BV or controls (p<0.05). Thus, we have developed a TSC-based xenograft mouse model that allows us to study IA chemotherapy. However, further studies are needed to analyze the treatment effects after IA BV to assess tumor progression and overall animal survival.