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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. Keywords: gene therapy, AAV vector, non-human primates, intrathecal infusion, cisterna magna, lumbar, intracerebroventricular, pharmacokinetics, magnetic resonance imaging, cerebrospinal fluid

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.

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 theheparan 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.

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. © 2019 The Authors., This work was supported by Wylder Nation Foundation and by grants from Spanish Ministry of Economy and Competitivity (SAF-2014-57539-R and SAF2017-87698-R) to M.D.L. and from NIH-NINDS (R01NS073940) to K.S.B. A.P-C was a recipient of the FPU pre-doctoral fellowship from the Spanish Ministry of Economy and Competitivity. L.S. was a recipient of the Edward H. Schuchman Research Fellowship from the National Niemann-Pick Disease Foundation. E.H.S. and X.H. were supported by a MERIT award from the NIH (R37 HD28607) to E.H.S.

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.

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; Eng 2f/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; Eng 2f/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 P 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.

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.

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.

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.

In the present study, we compared the therapeutic effect of tumor-selective retroviral replicating vectors (RRV) expressing the yeast cytosine deaminase (CD) delivered by convection-enhanced delivery (CED) or simple injection, followed by systemic administration of the pro-drug, 5-fluorocytosine (5-FC). Treatment with RRV-CD and systemic 5-FC significantly increased survival in rodent U87MG glioma model in comparison with controls (P0.01). Interestingly, CED of RRV-CD followed by 5-FC further enhanced survival in this animal model in comparison with intra-tumoral injection of RRV-CD, followed by systemic 5-FC (P0.05). High expression levels of Ki-67 were found in untreated tumors compared with treated. Untreated tumors were also much larger than treated. CED resulted in excellent distribution of RRV while only partial distribution of RRV was obtained after injection. Furthermore, RRV-CD and CD were also found in tumors from treated rats at study end points. These results demonstrated that RRV vectors may efficiently transduce and stably propagate in malignant human glioma, thereby achieving a significant in situ amplification effect after initial administration. We conclude that delivery of RRV into the glioma by CED provides much wider vector distribution than simple injection, and this correlated with better therapeutic outcomes.

Protein aggregation as a result of misfolding is a common theme underlying neurodegenerative diseases. Accordingly, most recent studies aim to prevent protein misfolding and/or aggregation as a strategy to treat these pathologies. For instance, state-of-the-art approaches, such as silencing protein overexpression by means of RNA interference, are being tested with positive outcomes in preclinical models of animals overexpressing the corresponding protein. Therapies designed to treat central nervous system diseases should provide accurate delivery of the therapeutic agent and long-term or chronic expression by means of a nontoxic delivery vehicle. After several years of technical advances and optimization, gene therapy emerges as a promising approach able to fulfill those requirements. In this review we will summarize the latest improvements achieved in gene therapy for central nervous system diseases associated with protein misfolding (e.g., amyotrophic lateral sclerosis, Alzheimer’s, Parkinson’s, Huntington’s, and prion diseases), as well as the most recent approaches in this field to treat these pathologies.

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.

There is considerable interest in the use of adeno-associated virus serotype 9 (AAV9) for neurological gene therapy partly because of its ability to cross the blood-brain barrier to transduce astrocytes and neurons. This raises the possibility that AAV9 might also transduce antigen-presenting cells (APC) in the brain and provoke an adaptive immune response. We tested this hypothesis by infusing AAV9 vectors encoding foreign antigens, namely human aromatic L-amino acid decarboxylase (hAADC) and green fluorescent protein (GFP), into rat brain parenchyma. Over ensuing weeks, both vectors elicited a prominent inflammation in transduced brain regions associated with upregulation of MHC II in glia and associated lymphocytic infiltration. Transduction of either thalamus or striatum with AAV9-hAADC evinced a significant loss of neurons and induction of anti-hAADC antibodies. We conclude that AAV9 transduces APC in the brain and, depending on the immunogenicity of the transgene, can provoke a full immune response that mediates significant brain pathology. We emphasize, however, that these observations do not preclude the use of AAV serotypes that can transduce APC. However, it does potentially complicate preclinical toxicology studies in which non-self proteins are expressed at a level sufficient to trigger cell-mediated and humoral immune responses.

Delivery of neurotrophic factors to treat neurodegenerative diseases has not been efficacious in clinical trials despite their known potency for promoting neuronal growth and survival. Direct gene delivery to the brain offers an approach for establishing sustained expression of neurotrophic factors but is dependent on accurate surgical procedures to target specific anatomical regions of the brain. Serotype-2 adeno-associated viral (AAV2) vectors have been investigated in multiple clinical studies for neurological diseases without adverse effects, however the absence of significant clinical efficacy after neurotrophic factor gene transfer has been largely attributed to insufficient coverage of the target region. Our pre-clinical development of AAV2-glial-derived neurotrophic factor (GDNF) for Parkinson’s disease involved real-time image guided delivery and optimization of delivery techniques to maximize gene transfer in the putamen. We have demonstrated that AAV2 vectors are anterogradely transported in the primate brain with GDNF expression observed in the substantia nigra after putaminal delivery in both intact and nigrostriatal lesioned primates. Direct midbrain delivery of AAV2-GDNF resulted in extensive anterograde transport to multiple brain regions and significant weight loss.

Background & Purpose: Brain arteriovenous malformation (bAVM) is a risk factor for intracranial hemorrhage. Current therapies are associated with high morbidities. We tested a minimally invasive gene therapy using an adeno-associated viral vector (AAV9) to intravenously (IV) deliver an anti-angiogenic agent, soluble FLT1 (sFLT1), to two bAVM mouse models. Method: Model 1 consists of SM22α Cre; Endoglin (Eng, a bAVM causative gene)-floxed mice, which have a 90% occurrence of spontaneous bAVM after 5 weeks and 50% mortality after 6 weeks. In Model 2, bAVM is induced in RosaCreER; Eng -floxed mice through tamoxifen-mediated Eng -deletion and AAV1-VEGF-induced brain angiogenesis. AAV9-sFLT1 or AAV9-GFP was IV injected to 4-week-old Model 1 mice, or Model 2 mice 8 weeks after model induction. Brain vasculature was analyzed 4 weeks later through vessel-casting and brain section staining with CD31 antibody. Potential toxicities were assessed by analyzing brain and liver histology, and measuring serum alkaline phosphatase (ALP) and alanine transaminase (ALT) activities as well as creatinine (CR) levels. Results: sFLT1-treatment increased Model 1 mice survival. BAVMs were detected in all GFP-treated, but only in 25% of sFLT1-treated mice. In Model 2, sFLT1 treatment reduced abnormal vessels. No neuronal death or lymphocyte was detected in the brain of sFLT1-treated mice. AAV9 treatment did not cause weight loss, or alter CR levels or ALP and ALT activities. Small clusters of inflammatory cells were detected in the liver of 58% of GFP- and 56% of sFLT1-treated mice. Conclusion: Systemic sFLT1 gene therapy reduces bAVM severity with minimal adverse effect. Future studies should consider restricting sFLT1 expression in the brain to reduce liver inflammation.

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.

The use of viral vectors to express therapeutics genes in Parkinson’s disease (PD) trials has been hindered by a lack of understanding of the principles that guide effective distribution of vectors within the basal ganglia, even when we have a strong expectation of efficacy based on experimentation in animal models. The major problems we have faced include (1) scale-up from small rodent and nonhuman primates (NHP) brains to humans, (2) understanding how viral vectors distribute within the brain parenchyma, (3) prediction of how viral particles are disseminated by neuronal projections after direct delivery to the putamen and substantia nigra, (4) the mechanism of action of the therapeutic gene on dopaminergic system, and (5) the relevance of animal models to idiopathic PD. In this chapter, we will address these important issues and will try to put them into the context of data that has been obtained from current and recent clinical trials. In particular, we will address therapeutic strategies aimed at restoring dopaminergic function by either expressing genes that encode enzymes responsible for synthesis of dopamine (DA) or expressing growth factors capable of upregulating DA function in the degenerated neurons. We will not address inhibition of outflow innervation from the striatum in PD patients by expressing glutamic acid decarboxylase (GAD) as this strategy is described in the dedicated chapter in this book.

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.

Niemann-Pick disease Type A (NPA) is a neuronopathic lysosomal storage disease (LSD) caused by the loss of acid sphingomyelinase (ASM). The goals of the current study are to ascertain the levels of human ASM that are efficacious in ASM knockout (ASMKO) mice, and determine whether these levels can be attained in non-human primates (NHPs) using a multiple parenchymal injection strategy. Intracranial injections of different doses of AAV1-hASM in ASMKO mice demonstrated that only a small amount of enzyme (10 mg hASM/g tissue was reached. In monkeys, injection of 12 tracts of AAV1-hASM resulted in efficacious levels of enzyme in broad regions of the brain that was aided, in part, by axonal transport of adeno-associated virus (AAV) and movement through the perivascular space. This study demonstrates that a combination cortical, subcortical, and cerebellar injection protocol could provide therapeutic levels of hASM to regions of the NHP brain that are highly affected in NPA patients. The information from this study might help design new AAV-mediated enzyme replacement protocols for NPA and other neuronopathic LSDs in future clinical trials.

In this review, we discuss recent developments in the delivery of adeno-associated virus-based vectors (AAV), particularly with respect to the role of axonal transport in vector distribution in the brain. The use of MRI-guidance and new stereotactic aiming devices have now established a strong foundation for neurological gene therapy to become an accepted procedure in interventional neurology.

We report the results of a long-term follow-up of subjects in a phase 1 study of AAV2-hAADC (adeno-associated virus type 2–human aromatic L-amino acid decarboxylase) gene therapy for the treatment of Parkinson's disease (PD). Ten patients with moderately advanced PD received bilateral putaminal infusions of either a low or a high dose of AAV2-hAADC vector. An annual positron emission tomography (PET) imaging with [18F]fluoro-L-m-tyrosine tracer was used for evaluation of AADC expression, and a standard clinical rating scale [Unified Parkinson's Disease Rating Scale (UPDRS)] was used to assess effect. Our previous analysis of the 6-month data suggested that this treatment was acutely safe and well tolerated. We found that the elevated PET signal observed in the first 12 months persisted over 4 years in both dose groups. A significantly increased PET value compared with the presurgery baseline was maintained over the 4-year monitoring period. The UPDRS in all patients off medication for 12 hr improved in the first 12 months, but displayed a slow deterioration in subsequent years. This analysis demonstrates that apparent efficacy continues through later years with an acceptable safety profile. These data indicate stable transgene expression over 4 years after vector delivery and continued safety, but emphasize the need for a controlled efficacy trial and the use of a higher vector dose.

We have previously shown that adeno-associated virus type 2 (AAV2) undergoes anterograde axonal transport in rat and non-human primate brain. We screened other AAV serotypes for axonal transport and found that AAV6 is transported almost exclusively in a retrograde direction and, in the same way as AAV2, it is also neuron-specific in rat brain. Our findings show that axonal transport of AAV is serotype dependent and this has implications for gene therapy of neurological diseases such as Huntington's disease.

Degeneration of nigrostriatal neurons in Parkinson's disease (PD) causes progressive loss of aromatic l-amino acid decarboxylase (AADC), the enzyme that converts levodopa (l-DOPA) into dopamine in the striatum. Because loss of this enzyme appears to be a major driver of progressive impairment of response to the mainstay drug, l-DOPA, one promising approach has been to use gene therapy to restore AADC activity in the human putamen and thereby restore normal l-DOPA response in patients with PD. An open-label phase I clinical trial of this approach in patients with PD provided encouraging signs of improvement in Unified Parkinson's Disease Rating Scale scores and reductions in antiparkinsonian medications. However, such improvement was modest compared with the results previously reported in parkinsonian rhesus macaques. The reason for this discrepancy may have been that the relatively small volume of vector infused in the clinical study restricted the distribution of AADC expression, such that only about 20% of the postcommissural putamen was covered, as revealed by l-[3-(18)F]-α-methyltyrosine-positron emission tomography. To achieve more quantitative distribution of vector, we have developed a visual guidance system for parenchymal infusion of AAV2. The purpose of the present study was to evaluate the combined magnetic resonance imaging-guided delivery system with AAV2-hAADC under conditions that approximate the intended clinical protocol. Our data indicate that this approach directed accurate cannula placement and effective vector distribution without inducing any untoward effects in nonhuman primates infused with a high dose of AAV2-hAADC.

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.

A pilot study in nonhuman primates was conducted, in which two Rhesus macaques received bilateral parenchymal infusions of adeno-associated virus serotype 9 encoding green fluorescent protein (AAV9-GFP) into each putamen. The post-surgical in-life was restricted to 3 weeks in order to minimize immunotoxicity expected to arise from expression of GFP in antigen-presenting cells. Three main findings emerged from this work. First, the volume over which AAV9 expression was distributed (Ve) was substantially greater than the volume of distribution of MRI signal (Vd). This stands in contrast with Ve/Vd ratio of rAAV2, which is lower under similar conditions. Second, post-mortem analysis revealed expression of GFP in thalamic and cortical neurons as well as dopaminergic neurons projecting from substantia nigra pars compacta, indicating retrograde transport of AAV9. However, fibers in the substantia nigra pars reticulata, a region that receives projections from putamen, also stained for GFP, indicating anterograde transport of AAV9 as well. Finally, one hemisphere received a 10-fold lower dose of vector compared with the contralateral hemisphere (1.5 × 10(13) vg ml(-1)) and we observed a much stronger dose effect on anterograde-linked than on retrograde-linked structures. These data suggest that AAV9 can be axonally transported bi-directionally in the primate brain. This has obvious implications to the clinical developing of therapies for neurological disorders like Huntington's or Alzheimer's diseases.

Clinical trials involving direct infusion of neurotrophic therapies for Parkinson's disease (PD) have suffered from poor coverage of the putamen. The planned use of a novel interventional-magnetic resonance imaging (iMRI) targeting system for achieving precise, real-time convection-enhanced delivery in a planned clinical trial of adeno-associated virus serotype 2 (AAV2)-glial-derived neurotrophic factor (GDNF) in PD patients was modeled in nonhuman primates (NHP). NHP received bilateral coinfusions of gadoteridol (Gd)/AAV2-GDNF into two sites in each putamen, and three NHP received larger infusion volumes in the thalamus. The average targeting error for cannula tip placement in the putamen was

We elucidated the effects of parkinsonian degeneration on trafficking of AAV2-GDNF in the nigro-striatum (nigro-ST) of unilaterally 6-hydroxydopamine (6-OHDA)-lesioned rats. Vector infused into striatum (ST) was transported to substantia nigra (SN), both pars compacta (SNc), and pars reticulata (SNr). In the lesioned hemisphere, glial cell line–derived neurotrophic factor (GDNF) immunoreactivity was only found in SNr consistent with elimination of SNc dopaminergic (DA) neurons by 6-OHDA. Further analysis showed that striatal delivery of AAV2-GDNF resulted in GDNF expression in globus pallidus (GP), entopeduncular nucleus (EPN), and subthalamic nucleus (STN) in both lesioned and unlesioned hemispheres. Injection of vector into SN, covering both SNc and SNr, resulted in striatal expression of GDNF in the unlesioned hemisphere but not in the lesioned hemisphere. No expression was seen in GP or EPN. We conclude that adeno-associated virus serotype 2 (AAV2) is transported throughout the nigro-ST exclusively by anterograde transport. This transport phenomenon directs GDNF expression throughout the basal ganglia in regions that are adversely affected in Parkinson's disease (PD) in addition to SNc. Delivery of vector to SN, however, does not direct expression of GDNF in ST, EPN, or GP. On this basis, we believe that striatal delivery of AAV2-GDNF is the preferred course of action for trophic rescue of DA function.

Optimal results in the direct brain delivery of brain therapeutics such as growth factors or viral vector into primate brain depend on reproducible distribution throughout the target region. In the present study, we retrospectively analyzed MRI of 25 convection-enhanced delivery (CED) infusions with MRI contrast into the putamen of non-human primates (NHP). Infused volume ( V i ) was compared to total volume of distribution ( V d ) versus V d within the target putamen. Excellent distribution of contrast agent within the putamen was obtained in eight cases that were used to define an optimal target volume or “green” zone. Partial or poor distribution with leakage into adjacent anatomical structures was noted in 17 cases, defining “blue” and “red” zones, respectively. Quantitative containment (99 ± 1%) of infused gadoteridol within the putamen was obtained when the cannula was placed in the green zone, 87 ± 3% in the blue zone and 49 ± 0.05% in the red zone. These results were used to determine a set of 3D stereotactic coordinates that define an optimal site for putaminal infusions in NHP and human putamen. We conclude that cannula placement and definition of optimal (green zone) stereotactic coordinates have important implications in ensuring effective delivery of therapeutics into the putamen utilizing routine stereotactic MRI localization procedures and should be considered when local therapies such as gene transfer or protein administration are being translated into clinical therapy.

Gene replacement therapy for the neurological deficits caused by lysosomal storage disorders, such as in Niemann-Pick disease type A, will require widespread expression of efficacious levels of acid sphingomyelinase (ASM) in the infant human brain. At present there is no treatment available for this devastating pediatric condition. This is partly because of inherent constraints associated with the efficient delivery of therapeutic agents into the CNS of higher order models. In this study we used an adeno-associated virus type 2 (AAV2) vector encoding human acid sphingomyelinase tagged with a viral hemagglutinin epitope (AAV2-hASM-HA) to transduce highly interconnected CNS regions such as the brainstem and thalamus. On the basis of our data showing global cortical expression of a secreted reporter after thalamic delivery in nonhuman primates (NHPs), we set out to investigate whether such widespread expression could be enhanced after brainstem infusion. To maximize delivery of the therapeutic transgene throughout the CNS, we combined a single brainstem infusion with bilateral thalamic infusions in naive NHPs. We found that enzymatic augmentation in brainstem, thalamic, cortical, as well subcortical areas provided convincing evidence that much of the large NHP brain can be transduced with as few as three injection sites.

Object The purpose of this study was to optimize stereotactic coordinates for delivery of therapeutic agents into the thalamus and brainstem, using convection-enhanced delivery (CED) to avoid leakage into surrounding anatomical structures while maximizing CED of therapeutics within the target volume. Methods The authors recently published targeting data for the nonhuman primate putamen in which they defined infusion parameters, referred to as “red,” “blue,” and “green” zones, that describe cannula placements resulting in poor, suboptimal, and optimal volumes of distribution, respectively. In the present study, the authors retrospectively analyzed 22 MR images with gadoteridol as a contrast reagent, which were obtained during CED infusions into the thalamus (14 cases) and brainstem (8 cases) of nonhuman primates. Results Excellent distribution of gadoteridol within the thalamus was obtained in 8 cases and these were used to define an optimal target locus (or green zone). Good distribution in the thalamus, with variable leakage into adjacent anatomical structures, was noted in 6 cases, defining a blue zone. Quantitative containment (99.7 ± 0.2%) of gadoteridol within the thalamus was obtained when the cannula was placed in the green zone, and less containment (85.4 ± 3.8%) was achieved with cannula placement in the blue zone. Similarly, a green zone was also defined in the brainstem, and quantitative containment of infused gadoteridol within the brainstem was 99.4 ± 0.6% when the cannula was placed in the green zone. These results were used to determine a set of 3D stereotactic coordinates that define an optimal site for infusions intended to cover the thalamus and brainstem of nonhuman primates. Conclusions The present study provides quantitative analysis of cannula placement and infusate distribution using real-time MR imaging and defines an optimal zone for infusion in the nonhuman primate thalamus and brainstem. Cannula placement recommendations developed from such translational nonhuman primate studies have significant implications for the design of anticipated clinical trials featuring CED therapy into the thalamus and brainstem for CNS diseases.

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.

Objective—Our group has pioneered the use of gadolinium liposomes (GDL) in convectionenhanced delivery (CED) using real-time MRI (magnetic resonance imaging) to visualize the distribution of therapeutics in in non-human primate (NHP) and canine brain. We have shown that this procedure is highly predictable and safe. In the course of recent studies, however, we noted that infusion of large volumes caused local anatomical alterations, such as ventricular compression, to occur. This study reports our analysis of CED infusions into normal brains and those compromised by tumors and how monitoring the CED infusion with MRI may be helpful in preventing some complications. Methods—A total of fifty-four CED infusions using gadolinium liposomes (GDL) were performed in seven canines and ten NHPs, and monitored using real-time MRI. The canines, harboring brain tumors, received infusions of GDL as well as a chemotherapeutic agent via CED. The NHPs were normal and received GDL infusions alone. Real-time analysis of the CED infusion was carried looking for proper catheter position, and infusion reflux, leakage, and mass effect. Retrospective analysis allowed assessment of CED volume of distribution versus volume of infusion. Results—Approximately ten percent of these infusions caused anatomical compression of the ventricles, especially in the canines with tumors. Reflux along the cannula and leakage of infusate into the ventricular CSF or subarachnoid space was seen. Animal behavior, however, did not appear to be affected acutely or during the time course of the study, and no ventricular compression was noted two weeks after the CED infusion on further brain imaging studies. Conclusion—These findings illustrate the value of being able to monitor infusions with real time MRI in order to identify phenomena such as reflux along the cannula, leakage of infusate, and ventricular compression. Especially in tumor patients, the latter could be associated with morbidity.

We are developing a method for real-time magnetic resonance imaging (MRI) visualization of convection-enhanced delivery (CED) of adeno-associated viral vectors (AAV) to the primate brain. By including gadolinium-loaded liposomes (GDL) with AAV, we can track the convective movement of viral particles by continuous monitoring of distribution of surrogate GDL. In order to validate this approach, we infused two AAV (AAV1-GFP and AAV2-hAADC) into three different regions of non-human primate brain (corona radiata, putamen, and thalamus). The procedure was tolerated well by all three animals in the study. The distribution of GFP determined by immunohistochemistry in both brain regions correlated closely with distribution of GDL determined by MRI. Co-distribution was weaker with AAV2-hAADC, although in vivo PET scanning with FMT for AADC activity correlated well with immunohistochemistry of AADC. Although this is a relatively small study, it appears that AAV1 correlates better with MRI-monitored delivery than does AAV2. It seems likely that the difference in distribution may be due to differences in tissue specificity of the two serotypes.

We investigated the safety and neuroregenerative potential of an adeno-associated virus (AAV2) containing human glial cell line-derived neurotrophic factor (GDNF) in an MPTP primate model of Parkinson's disease. Dopaminergic function was evaluated by positron emission tomography with 6-[(18)F]fluoro-l-m-tyrosine (FMT) before and after AAV2-GDNF or phosphate-buffered saline infusion bilaterally into the putamen. FMT uptake was significantly increased bilaterally in the putamen of AAV2-GDNF but not phosphate-buffered saline-treated animals 6 months after infusion, indicating increased dopaminergic activity in the nigrostriatal pathways. AAV2-GDNF-treated animals also showed clinical improvement without adverse effects. These findings are consistent with our previous report in aged nonhuman primates that showed evidence of enhanced use of striatal dopamine and dopaminergic nigrostriatal innervation. Clinical improvement and evidence of functional recovery in the nigrostriatal pathway, and the absence of adverse effects, support the safety of this approach for the delivery of GDNF over a 6-month period.

Growth factor therapy for Parkinson's disease offers the prospect of restoration of dopaminergic innervation and/or prevention of neurodegeneration. Safety and efficacy of an adeno-associated virus (AAV2) encoding human glial cell-derived neurotrophic factor (GDNF) was investigated in aged nonhuman primates. Positron emission tomography with 6-[(18)F]-fluoro-l-m-tyrosine (FMT-PET) in putamen was assessed 3 months before and after AAV2 infusion. In the right putamen, monkeys received either phosphate-buffered saline or low-dose (LD) or high-dose (HD) AAV2-GDNF. Monkeys that had received putaminal phosphate-buffered saline (PBS) infusions additionally received either PBS or HD AAV2-GDNF in the right substantia nigra (SN). The convection-enhanced delivery method used for infusion of AAV2-GDNF vector resulted in robust volume of GDNF distribution within the putamen. AAV2-GDNF increased FMT-PET uptake in the ipsilateral putamen as well as enhancing locomotor activity. Within the putamen and caudate, the HD gene transfer mediated intense GDNF fiber and extracellular immunoreactivity (IR). Retrograde and anterograde transport of GDNF to other brain regions was observed. AAV2-GDNF did not significantly affect dopamine in the ipsilateral putamen or caudate, but increased dopamine turnover in HD groups. HD putamen treatment increased the density of dopaminergic terminals in these regions. HD treatments, irrespective of the site of infusion, increased the number of nonpigmented TH-IR neurons in the SN. AAV2-GDNF gene transfer does not appear to elicit adverse effects, delivers therapeutic levels of GDNF within target brain areas, and enhances utilization of striatal dopamine and dopaminergic nigrostriatal innervation.

Transduction of the primate cortex with adeno-associated virus (AAV)-based gene therapy vectors has been challenging, because of the large size of the cortex. We report that a single infusion of AAV2 vector into thalamus results in widespread expression of transgene in the cortex through transduction of widely dispersed thalamocortical projections. This finding has important implications for the treatment of certain genetic and neurodegenerative diseases.

In Parkinson's disease (PD) chronic inflammation occurs in the substantia nigra (SNc) concurrently with dopaminergic neurodegeneration. In models of PD, microglial activation precedes neurodegeneration in the SNc, suggesting that the underlying pathogenesis involves a complex response in the nigrostriatal pathway, and that the innate immune system plays a significant role. We have investigated the neuroprotective effect of an adeno-associated viral type-2 (AAV2) vector containing the complementary DNA (cDNA) for human interleukin-10 (hIL-10) in the unilateral 6-hydroxydopamine (6-OHDA) rat model of PD. AAV2-hIL-10 reduced the 6-OHDA-induced loss of tyrosine hydroxylase (TH)-positive neurons in the SNc, and also reduced loss of striatal dopamine (DA). Pretreatment with AAV2-hIL-10 reduced glial activation in the SNc but did not attenuate striatal release of the inflammatory cytokine IL-1beta. Assessment of rotational behavior in response to apomorphine challenge showed absence of asymmetry, confirming protection of dopaminergic innervation of the lesioned striatum. At baseline, 6-OHDA-lesioned animals displayed a deficit in contralateral forelimb use, but pretreatment with AAV2-hIL-10 reduced this forelimb akinesia. Transcriptional analyses revealed alteration of a few genes by AAV2-hIL-10; these alterations may contribute to neuroprotection. This study supports the need for further investigations relating to gene therapies aimed at reducing neuroinflammation in early PD.

The incidence of Parkinson's disease (PD) increases greatly with age, and the baby-boomer population can expect to generate a large number of individuals with the disease, all of whom will have significantly increased medical care needs over periods of 20 years or more. This emerging healthcare burden to our society calls for accelerated efforts to understand this disease better and treat it more effectively. The growing interest in gene therapy grew out of a recognition that new medicines may be needed to combat the relentless progression of the disease in the face of conventional pharmaco-therapies and surgical interventions that have so far failed to offer more than palliative relief. The potential of gene therapy to alter dramatically the course of the disease lies very much with the challenge of converting a research tool into a medical option, a process that clearly requires a unique combination of rigor and flexibility. In this review, we examine the unique aspects of gene therapy that make its use in PD attractive, but also analyze the difficulties of employing a medicine that acts for the rest of the patient's life.

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.

Adeno-associated virus serotype 2 (AAV2) has previously been reported to be a slowly uncoating virus in peripheral tissues, but persistence of intact vector in primate brain has not been explored. Because some neurological gene therapies may require re-administration of the same vector to patients, it seems important to understand the optimal timeframe in which to consider such repeat intervention. Surprisingly, convection-enhanced delivery of AAV2 into the thalamus of nonhuman primates (NHPs) resulted in robust staining of neurons with A20 antibody that detected intact AAV2 particles at ∼1.5 months after infusion. However, by 2.5 months, no A20 staining was visible. These data confirmed earlier findings of persistence of intact AAV2 particles in ocular and hepatic tissues. In order to probe the potential consequences of this persistence, we infused AAV2-human aromatic L-amino acid decarboxylase into left and right thalamus of three NHPs, with a 3-month delay between infusions. During that interval, we immunized each animal subcutaneously with AAV2 virus-like particles (empty vector) in order to induce strong anti-capsid humoral immunity. Various high neutralizing antibody titers were achieved. The lowest titer animal showed infiltration of B lymphocytes and CD8(+) T cells into both the secondary and primary infusion sites. In the other two animals, extremely high titers resulted in no transduction of the second site and, therefore, no lymphocytic infiltration. However, such infiltration was prominent at the primary infusion site in each animal and was associated with overt neuronal loss and inflammation.