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

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

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

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

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.

Dynamic hyperpolarized carbon-13 MR metabolic imaging of nonhuman primate brain.

PURPOSE: To investigate hyperpolarized (13) C metabolic imaging methods in the primate brain that can be translated into future clinical trials for patients with brain cancer. METHODS: (13) C coils and pulse sequences designed for use in humans were tested in phantoms. Dynamic (13) C data were obtained from a healthy cynomolgus monkey brain using the optimized (13) C coils and pulse sequences. The metabolite kinetics were estimated from two-dimensional localized (13) C dynamic imaging data from the nonhuman primate brain. RESULTS: Pyruvate and lactate signal were observed in both the brain and the surrounding tissues with the maximum signal-to-noise ratio of 218 and 29 for pyruvate and lactate, respectively. Apparent rate constants for the conversion of pyruvate to lactate and the ratio of lactate to pyruvate showed a difference between brain and surrounding tissues. CONCLUSION: The feasibility of using hyperpolarized [1-(13) C]-pyruvate for assessing in vivo metabolism in a healthy nonhuman primate brain was demonstrated using a hyperpolarized (13) C imaging experimental setup designed for studying patients with brain tumors. The kinetics of the metabolite conversion suggests that this approach may be useful in future studies of human neuropathology.

Safety and tolerability of magnetic resonance imaging-guided convection-enhanced delivery of AAV2-hAADC with a novel delivery platform in nonhuman primate striatum.

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.

Regeneration of the MPTP-lesioned dopaminergic system after convection-enhanced delivery of AAV2-GDNF.

Clinical studies to date have failed to establish therapeutic benefit of glial cell-derived neurotrophic factor (GDNF) in Parkinson's disease (PD). In contrast to previous nonclinical neuroprotective reports, this study shows clinically relevant and long-lasting regeneration of the dopaminergic system in rhesus macaques lesioned with 1-methy-4-phenyl-1,2,3,6-tetrahydropyridine 3-6 months before GDNF gene delivery (AAV2-GDNF). The observed progressive amelioration of functional deficits, recovery of dopamine, and regrowth of fibers to the striatal neuropil demonstrate that high GDNF expression in the putamen promotes restoration of the dopaminergic system in a primate model of advanced PD. Extensive distribution of GDNF within the putamen and transport to the severely lesioned substantia nigra, after convection-enhanced delivery of AAV2-GDNF into the putamen, indicates anterograde transport via striatonigral connections and is anticipated to occur in PD patients. Overall, these data demonstrate nonclinical neurorestoration after putaminal infusion of AAV2-GDNF and suggest that clinical investigation in PD patients is warranted.

Eight years of clinical improvement in MPTP-lesioned primates after gene therapy with AAV2-hAADC.

This study completes the longest known in vivo monitoring of adeno-associated virus (AAV)-mediated gene expression in nonhuman primate (NHP) brain. Although six of the eight parkinsonian NHP originally on study have undergone postmortem analysis, as described previously, we monitored the remaining two animals for a total of 8 years. In this study, NHP received AAV2-human L-amino acid decarboxylase (hAADC) infusions into the MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned putamen. Restoration of AADC activity restored normal response to levodopa and gene expression could be quantitated repeatedly over many years by 6-[(18)F]fluoro-meta-tyrosine (FMT)-positron emission tomography (PET) and confirm that AADC transgene expression remained unchanged at the 8-year point. Behavioral assessments confirmed continued, normalized response to levodopa (improvement by 35% over historical controls). Postmortem analysis showed that, although only 5.6 + or - 1% and 6.6 + or - 1% of neurons within the transduced volumes of the striatum were transduced, this still secured robust clinical improvement. Importantly, there were no signs of neuroinflammation or reactive gliosis at the 8-year point, indicative of the safety of this treatment. The present data suggest that the improvement in the L-3,4-dihydroxyphenylalanine (L-Dopa) therapeutic window brought about by AADC gene therapy is pronounced and persistent for many years.

Real-time MR imaging with Gadoteridol predicts distribution of transgenes after convection-enhanced delivery of AAV2 vectors.

Gene therapies that utilize convention-enhanced delivery (CED) will require close monitoring of vector infusion in real time and accurate prediction of drug distribution. The magnetic resonance imaging (MRI) contrast agent, Gadoteridol (Gd), was used to monitor CED infusion and to predict the expression pattern of glial cell line-derived neurotrophic factor (GDNF) protein after administration of adeno-associated virus type 2 (AAV2) vector encoding human pre-pro-GDNF complementary DNA. The nonhuman primate (NHP) thalamus was utilized for modeling infusion to allow delivery of volumes more relevant to planned human studies. AAV2 encoding human aromatic L-amino acid decarboxylase (AADC) was coinfused with AAV2-GDNF/Gd to confirm regions of AAV2 transduction versus extracellular GDNF diffusion. There was a close correlation between Gd distribution and GDNF or AADC expression, and the ratios of expression areas of GDNF or AADC versus Gd were both close to 1. Our data support the use of Gd and MRI to monitor AAV2 infusion via CED and to predict the distribution of GDNF protein after AAV2-GDNF administration.

Safety evaluation of AAV2-GDNF gene transfer into the dopaminergic nigrostriatal pathway in aged and parkinsonian rhesus monkeys.

We evaluated neuropathological findings in two studies of AAV2-GDNF efficacy and safety in naive aged (>20 years) or MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine)-lesioned rhesus macaques. In the first study, a total of 17 animals received one of two doses of AAV2-GDNF into either putamen or substantia nigra (SN). To control for surgical variables, all animals received identical putaminal and nigral infusions in which phosphate-buffered saline was substituted for vector as appropriate. All 17 aged monkeys were studied for 6 months before necropsy. In a separate study, 11 MPTP-lesioned rhesus macaques with extensive lesions in the right SN and mild lesions in the left SN received bilateral infusions of AAV2-GDNF (9.9 x 10(11) vector genomes) or PBS into the putamen and were then studied for up to 14 months. In the current analysis, we addressed safety issues regarding AAV2-GDNF administration. An extensive series of assessments of in-life behavioral and clinical parameters was conducted. No overt histopathology or immune responses were detected in any experimental monkey. However, the delivery of AAV2-GDNF to the SN of aged monkeys caused a marked and significant loss of body weight (-19.4%). No weight loss was observed in the MPTP-lesioned monkeys despite bilateral axonal transport of glial cell line-derived neurotrophic factor (GDNF) to the SN from the putamen. These findings indicate that putaminal administration of AAV2-GDNF by convection-enhanced delivery shows therapeutic promise without any apparent side effects. Importantly, nigral administration of AAV2-GDNF caused significant weight loss that raises substantial concern for clinical application of this approach.

Functional effects of AAV2-GDNF on the dopaminergic nigrostriatal pathway in parkinsonian rhesus monkeys.

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.

Clinically relevant effects of convection-enhanced delivery of AAV2-GDNF on the dopaminergic nigrostriatal pathway in aged rhesus monkeys.

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.

Biodistribution of adeno-associated virus type-2 in nonhuman primates after convection-enhanced delivery to brain.

A combination treatment of AAV2-hAADC with oral levodopa is a novel therapeutic approach that is being developed for late-stage Parkinson's disease. Biodistribution of AAV2-hAADC was assessed over a wide range of vector dose in 12 monkeys with parkinsonian syndrome, 6 months after intraputamenal infusion. Quantitative PCR (Q-PCR) from all the major neuroanatomical regions of the brain indicated a dose-dependent increase in vector DNA, with 99% being detected in the target site and other basal ganglia tissues. Within these tissues, the distribution varied widely between the putamen (PT) and the globus pallidus, and this was attributed to differences in vector transport. Q-PCR and immunocytochemistry were consistent with results reported earlier for various measures of transgene expression including aromatic L-amino acid decarboxylase (AADC) activity assays, behavioral response, and in vivo imaging with positron emission tomography (PET). Outside of the brain, trace amounts of vector DNA were detected in the spleens of animals in the two highest dose groups, but not in any other peripheral tissue, blood, or cerebrospinal fluid. Some increase in neutralizing antibody titers to adeno-associated virus type-2 (AAV2) capsid protein was observed in monkeys that received high doses of AAV2-hAADC or control AAV2-GFP. This study further validates convection-enhanced delivery (CED) as the preferred method of viral vector delivery to the brain, and supports a Phase I clinical testing of AAV2-hAADC in humans with Parkinson's disease.

Long-term clinical improvement in MPTP-lesioned primates after gene therapy with AAV-hAADC.

Dopamine, the major neurotransmitter depleted in Parkinson disease, can be synthesized and regulated in vivo with a combination of intrastriatal AAV-hAADC gene therapy and administration of the dopamine precursor l-Dopa. When tested in MPTP-lesioned monkeys, this approach resulted in long-term improvement in clinical rating scores, significantly lowered l-Dopa requirements, and a reduction in l-Dopa-induced side effects. Positron emission tomography with [(18)F]FMT confirmed persistent AADC activity, demonstrating for the first time that infusion of AAV vector into primate brain results in at least 6 years of transgene expression. AAV-hAADC restores the ability of the striatum to convert l-Dopa into dopamine efficiently. Introduction of this therapy into the clinic holds promise for Parkinson patients experiencing the motor complications that result from escalating l-Dopa requirements against a background of disease progression.

A dose-ranging study of AAV-hAADC therapy in Parkinsonian monkeys.

The main medication for idiopathic Parkinson disease is L-Dopa. Drug efficacy declines steadily in part because the converting enzyme, aromatic L-amino acid decarboxylase (AADC), is lost concomitant with substantia nigra atrophy. Over the past decade, we have developed a gene therapy approach in which AADC activity is restored to the brain by infusion into the striatum of a recombinant adeno-associated virus carrying human AADC cDNA. We report here the results of an investigation of the relationship between vector dose and a series of efficacy markers, such as PET, L-Dopa response, and AADC enzymatic activity. At low doses of vector, no effect of vector was seen on PET or behavioral response. At higher doses, a sharp improvement in both parameters was observed, resulting in an approximate 50% improvement in L-Dopa responsiveness. The relationship between vector dose and AADC enzymatic activity in tissue extracts was linear. We conclude that little behavioral improvement can be seen until AADC activity reaches a level that is no longer rate limiting for conversion of clinical doses of L-Dopa into dopamine or for trapping of the PET tracer FMT. These findings have implications for the design and interpretation of clinical studies of AAV-hAADC gene therapy.

Focal striatal dopamine may potentiate dyskinesias in parkinsonian monkeys.

Striatal neurons convert L-dopa to dopamine (DA) following gene transfer of aromatic L-amino acid decarboxylase (AADC) via adeno-associated virus (AAV) in parkinsonian monkeys. We investigated whether AAV-AADC could reduce or eliminate L-dopa-induced dyskinesias (LIDs) and side effects in MPTP-treated monkeys. Five monkeys were made parkinsonian by bilateral MPTP lesions. The optimal therapeutic dose of L-dopa was determined using an acute dose response regimen. After 3 weeks of chronic L-dopa treatment, AAV-AADC or control vector was bilaterally injected into the striatum. Animals were assessed for 6 months with the same L-dopa dosing as presurgery as well as chronic oral L-dopa treatment. Presurgery LID was observed at doses greater than 5 mg/kg. The AAV-AADC-treated animals displayed an average 7.3-fold decrease in the therapeutic dose of L-dopa throughout the 6-month follow-up period. Only AAV-AADC-treated monkeys were susceptible to dyskinesias even at sub-clinical doses. Immunohistochemical analysis revealed well-delineated foci of AADC within the striatum. These results suggest that high levels of focal DA were generated in response to L-dopa administration and may be responsible for the exacerbation of dyskinesias. This may be similar to focal dopaminergic activity in PD patients that developed off-drug or "runaway" dyskinesias following fetal mesencephalic grafts.